SciScore for 10.1101/2020.09.27.316174: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Hamster challenge experiments: Male and female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody binding ELISA: The S1 subunit of the SARS-CoV-2 spike protein (amino acids 16-685) bearing a C-terminal histidine tag (ACRO Biosystems, Newark, NJ) was coated at 2 μg/ml on a Ni-NTA plate (Qiagen, Valencia, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 spike protein (amino acids 16-685</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody characterization: Kinetic interactions between the antibodies and His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJ) protein was measured at 25°C using BIAcore T200 surface plasmon resonance (SPR) (GE Healthcare).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJ</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">STI-1499 or STI-2020 antibody was covalently immobilized on a CM5 sensor chip to approximately 500 and 100 resonance units (RU), respectively using standard N-hydroxysuccinimide/N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS/EDC) coupling methodology.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STI-2020</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For antibody binding to the cells expressing the Spike proteins, the cells were dispensed into wells of a 96-well plate (25 μl per well), and an equal volume of 2x final concentration of serially-diluted anti-S1 antibody solution was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-S1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody treatments were administered intravenously (i.v.) with monoclonal antibodies (mAbs) against SARS-CoV-2 Spike, or isotype control mAb in up to 350 µl of sterile PBS at 1 hour-post inoculation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 cells were transfected using FuGeneHD transfection reagent according to manufacturer’s protocol (Promega, Cat # E2311).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 cells were plated to 96-well plates and incubated at 37° C, 5% CO2</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell based Spike binding assay: Mammalian expression vectors were constructed either by cloning of the synthesized gene encoding SARS-CoV-2 G614 Spike protein (UniprotKB, SPIKE-SARS2) or, for SARS-CoV-2 D614 Spike protein, via site-directed mutagenesis of the G614 Spike protein gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniprotKB</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A sigmoidal four-parameter logistic equation was used for fitting the MFI vs. mAb concentration data set to extract EC50 values (GraphPad Prism 8.3.0 software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from rtransparent:

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

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Fascinating how human minds are almost like computers themselves.

This is very popular on social media, comical videos surrounding the internet, youtube creators that rely on the humor to help fuel their views

训练一个源头，只要某种类别的信息

DOI: 10.1074/jbc.RA120.015400

Resource: (Abcam Cat# ab1187, RRID:AB_298652)

Curator: @Naa003

SciCrunch record: RRID:AB_298652

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What is this?

<small><cite class='h-cite via'>ᔥ <span class='p-author h-card'>Cory Doctorow</span> in Pluralistic: 16 Feb 2021 – Pluralistic: Daily links (<time class='dt-published'>02/25/2021 12:20:24</time>)</cite></small>

It's interesting to note that there are already two other people who have used Hypothes and their page note functionality to tag this article as to read, one with (to read) and another with (TODO-read).

This is an excellent overview of how to take notes for academic research and creating writing output.

Others on the page here (specifically Dpthomas87's A, B, C) have done a great job at outlining their methods which I'm generally following. So I'll focus a bit more on the mechanics.

I rely pretty heavily on Hypothes.is for most of my note taking, highlights, and annotations. This works whether a paper is online or as a pdf I read online or store locally and annotate there.

Then I use RSS to pipe my data from Hypothes.is into a text file in OneDrive for my Obsidian vault using IFTTT.com. I know that a few are writing code for the Hypothes.is API to port data directly into Roam Research presently; I hope others might do it for Obsidian as well.)

Often at the end of the day or end of the week, I'll go through my drafts folder everything is in to review things, do some light formatting and add links, tags, or other meta data and links to related ideas.

Using Hypothes.is helps me get material into the system pretty quickly without a lot of transcription (which doesn't help my memory or retention). And the end of the day or end of week review helps reinforce things as well as help to surface other connections.

I'm hoping that as more people use Hypothesis for social annotation, the cross conversations will also be a source of more helpful cross-linking of ideas and thought.

I prefer to keep my notes as atomic as I can.

For some smaller self-contained things like lectures, I may keep a handful of notes together rather than splitting them apart, but they may be linked to larger structures like longer courses or topics of study.

If an article only has one or two annotations I'll keep them together in the same note, but books more often have dozens or hundreds of notes which I keep in separate files.

For those who don't have a clear idea of what or why they're doing this, I highly recommend reading [[Sönke Ahrens]]' book Smart Notes.

I do have a handful of templates for books, articles, and zettels to help in prompting me to fill in appropriate meta data for various notes more quickly. For this I'm using the built-in Templates plug-in and then ctrl-shift-T to choose a specific template as necessary.

Often I'll use Hypothes.is and tag things as #WantToRead to quickly bookmark things into my vault for later thought, reading, or processing.

For online videos and lectures, I'll often dump YouTube URLs into https://docdrop.org/, which then gives a side by side transcript for more easily jumping around as well as annotating directly from the transcript if I choose.

I prefer to use [[links]] over #tags for connecting information. Most of the tags I use tend to be for organizational or more personal purposes like #WantToRead which I later delete when done.

When I run across interesting questions or topics that would make good papers or areas of future research I'll use a tag like #OpenQuestion, so when I'm bored I can look at a list of what I might like to work on next.

Syndicated copies: https://forum.obsidian.md/t/research-phd-academics/1446/64?u=chrisaldrich

Common sense, so I'll tag as TYCO (Thank you, Captain Obvious).

argumentación

puntos que son un llamado a la acción, vinculado al propósito

citas, fuentes de consulta

DOI: 10.1016/j.nbd.2020.105141

Resource: (James Trimmer, University of California at Davis Cat# 12CA5, RRID:AB_2532070)

Curator: @Naa003

SciCrunch record: RRID:AB_2532070

Curator comments: Anti-HA tag mouse monoclonal antibody 12CA5 James Trimmer, University of California at Davis Cat# 12CA5

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What is this?

rare in computer science using category theory directly in computer science What other areas of math can be used / are rare to use directly in computer science?

In other words, you can build on this software in your proprietary software but can't change the Trailblazer source unless you're willing to contribute it back.

loophole: I wonder if this will actually just push people to move their code -- which at the core is/would be a direction modification to the source code - out to a separate module. That's so easy to do with Ruby, so this restriction hardly seems like it would have any effect on encouraging contributions.

Reviewer #2 (Public Review):

In this manuscript, the authors generated transgenic zebrafish reporter lines that allow observation of cytoplasmic lipid droplets in vivo. They knocked in GFP or RFP in the endogenous loci of perilipin 2 and 3, and showed that the reporter genes exhibited similar temporal and spatial expression in the intestine in response to acute high-fat feeding as the endogenous perilipin 2 and 3 transcripts. They also characterized the reporter gene expression in the liver, adipocytes, and around neuromasts. These tools open up new opportunities to study lipid droplets dynamics in live zebrafish that is not feasible in mouse models. Overall the manuscript is well written. The authors have discussed in details the strength and caveats of these reporters. The weakness is the descriptive nature of the study - many interesting observations but no mechanistic study. I have the additional comments:

1) It is curious that in plin2 and plin3 reporter fish, the fluorescent tags were inserted at the 5' and 3' of the open reading frame, respectively. The authors did not provide any explanation. Does the location where the fluorescent tag is inserted affect the expression of the reporter genes?

2) GFP and TagRFP-T are not fast folding fluorescent proteins and are very stable, which may not be the best options for studying the formation and degradation of lipid droplets. How the fluorescent tags affect the stability and clearance of the protein should be carefully characterized.

3) Was there any indel being introduced by TALENs in these knockin fish? Is there off target effects of the TALENs?

4) The authors also generated transgenic fish overexpressing human PLIN2 and PLIN3 fluorescent fusion proteins. Is the subcellular localization of these fusion proteins similar to the zebrafish knockin under nofed and fed conditions? In other words, do human PLIN2 and PLIN3 proteins behave similarly as the zebrafish orthologs?

This one I do not understands, likes can result in it becoming viral, retweets cause it to spread, why replies? You can tag specific people in the replies? Concern the person posting will be harassed? Where is the open dialogue to discuss the post more as encourage above?

NexusFont 是 Windows 下一款轻量好用的字体管理器。

NexusFont 不但能快速预览自定义的文本，还可以给字体打上 tag 进行分类，极大地提高了工作效率。

SciScore for 10.1101/2021.01.31.428824: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Four rounds of panning were used to isolate scFvs binding both MERS S2 and SARS-2 spike using the following solutions coated on high binding plates: 2 μg/ml anti-c-myc tag antibody (Invitrogen) to eliminate phage expressing no or truncated scFv (Round 1), 2 μg/ml MERS S2 (Round 2), 2 μg/ml SARS-2 spike (Round 3), and 0.4 μg/ml SARS-2 spike (Round 4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-c-myc tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Duplicate serial dilutions of each full-length antibody were allowed to bind each coat, and the secondary antibody solution was a 1:1200 dilution of goat-anti-human IgG Fc-HRP (SouthernBiotech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG Fc-HRP ( SouthernBiotech) .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western blot of antibody binding to coronavirus spike proteins: Purified coronavirus spike proteins (SARS-2 HexaPro, SARS-2, MERS, and HKU1) were reduced and boiled, and 50 ng of each was subjected to SDS-PAGE and transfer to PVDF membranes in quadruplicate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HKU1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine the affinity of 3A3 Fab by BLI, anti-human IgG Fc sensors were coated with the anti-foldon antibody identified in this work (3E11) at 20nM in kinetic buffer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-foldon</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MERS (18), HKU1 (18), and the SARS-2 variants HexaPro S2 (residues 697-1208 of the SARS-2 spike with an artificial signal peptide, proline substitutions at positions 817, 892, 899, 942, 986 and 987 and a C-terminal T4 fibritin domain, HRV3C cleavage site, 8xHisTag and TwinStrepTag), HexaPro RBD-locked-down (HexaPro with S383C-D985C substitutions), and aglycosylated HexaPro (HexaPro treated with Endo H overnight at 4 °C leaving only one N-acetylglucosamine attached to N-glycosylation site) as well as MERS S2-only (residues 763-1291 of MERS-2P with 8 additional stabilizing substitutions), MERS S2-apex-less (MERS S2-only construct with residues 811-824 replaced with GGSGGS and residues 1042-1073 replaced with a flexible linker) were expressed in Freestyle 293-F cells (ThermoFisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293-F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On day 2 after transfection, HEK-293T-hACE2 cells (BEI, NR-52511), which stably expresses human ACE2, were stained with 1 μM CellTrace Far Red dye (Invitrogen, Ex/Em: 630/661 nm) in PBS for 20 minutes at room temperature, then quenched with DMEM with 10% heat-inactivated FBS for 5 minutes, and resuspended in fresh media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T-hACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">expressing human ACE2 under an EF1a promoter was used to transduce HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry: On day 0, Expi-293 cells (ThermoFisher) were mock-transfected or transfected with pWT-SARS-2-spike (BEI NR-52514) or pD614G-SARS-2-spike (generated by site-directed mutagenesis).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi-293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Murine immunization: Three BALB/c mice were immunized subcutaneously with 5μg pre-fusion stabilized MERS S2 and 20 μg of ODN1826 + 100 μl of 2X Sigma Adjuvant System</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were collected with Zeiss LSM 710 confocal microscope (Carl Zeiss, Inc) and processed using ImageJ software (http://rsbweb.nih.gov/ij) (Fig. 2 and fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The statistical significance of either HEK-ACE2 colocalization percentage or average cell size between different conditions was calculated with ANOVA using GraphPad Prism 7 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spectra were manually assessed, and figures were prepared using HD-eXplosion (40) and PyMOL (41).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed again, then scanned for AF647 (640 nm excitation, 670/30 bandpass emission) fluorescence on a BD Fortessa flow cytometer and analyzed with FlowJo (Fig. 6B).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

There are several limitations to this work as currently described. First, a structure showing the atomic details of 3A3 complexed with spike would provide additional insight into the mechanism of binding and neutralization. However, structures of antibodies bound to S2 are generally challenging to obtain with just one structure available of an antibody binding near the HR2 stem (28). It is possible that 3A3 binding distorts spike structure, disturbing otherwise ordered regions. Accordingly, additional efforts to better understanding the molecular underpinnings of 3A3/ spike interactions are underway. Second, while we have shown that 3A3 binds spike from all three highly pathogenic coronaviruses with similar affinities, we have only demonstrated its ability to neutralize SARS-2 spikes in vitro. Demonstration of broad neutralization in addition to broad recognition would increase the potential relevance of this epitope for future therapeutics. The 3A3 epitope is highly conserved, with pairwise comparisons showing between 56% and 100% identity to the SARS-2 epitope for MERS and SARS-1, respectively (fig. S14). Since 3A3 affinity for the least similar MERS spike is comparable to that for the SARS-2 spike and greater than for HKU1, it seems likely that binding and neutralization depend primarily on RBD position epitope accessibility. The most concerning emerging SARS-2 variants have one conservative substitution in this epitope in B.1.1.7, identified in the United Kingdom, and has...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from scite Reference Check: We found one citation with an erratum. We recommend checking the erratum to confirm that it does not impact the accuracy of your citation.

<table style="border-collapse: collapse;"><tr><th style="min-width:95px; border: 1px solid lightgray; padding:2px">DOI</th><th style="min-width:95px; border: 1px solid lightgray; padding:2px">Status</th><th style="min-width:95px; border: 1px solid lightgray; padding:2px">Title</th></tr><tr><td style="min-width:95px; border: 1px solid lightgray; padding:2px">10.1371/journal.ppat.1000863</td><td style="min-width:95px; border: 1px solid lightgray; padding:2px">Has correction</td><td style="min-width:95px; border: 1px solid lightgray; padding:2px">In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methyla…</td></tr></table>

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Reviewer #3 (Public Review):

In this study from the Selimi lab, Gónzalez-Calvo and colleagues investigate the role of the uncharacterized complement family protein SUSD4. SUSD4 is expressed at the time of cerebellar synaptogenesis and localizes to dendritic spines of Purkinje cells. Susd4 KO mice show impaired motor learning, a cerebellum-dependent task. Susd4 KO mice display impaired LTD and facilitated LTP at parallel fiber (PF)-Purkinje cell (PC) synapses, indicating altered synaptic plasticity in the absence of Susd4. Climbing fiber (CF)-Purkinje cell synapses show largely normal basal transmission, with the exception of larger quantal EPSCs. Immunohistochemical analysis shows a small decrease in the proportion of CF/PC synapses lacking GluA2. As their data indicates a role for SUSD4 in regulation of postsynaptic GluA2 content at cerebellar synapses, they next explored the molecular mechanism by which SUSD4 might do so. Activity-dependent degradation of GluA2 does not occur in the absence of SUSD4. Affinity purification of proteins associated with recombinant SUSD4 identifies ubiquitin ligases as well as several proteins involved in AMPAR turnover. Finally, the authors show that SUSD4 can bind GluA2 in HEK cells, and that SUSD4 can bind the ubiquitin ligase NEDD4, but that these two interactions are not dependent on each other.

This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

Specific comments:

1) Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Fig. 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

2) Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

3) Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

4) Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

5) Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

6) Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

Pace Grandmaster Flash.

See annotations with the build-phases tag.

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Why are the build phases not enumerated in the Nix manual? If the instructions on how to create a derivation (and thus, a package) then why not go all in instead of spreading out information in different manuals, making the subject harder to grasp?...

(By the way, it is documented in the Nixpkgs manual under 6.5 Phases; not sure why it is not called build phases when every page refers to them like that.)

this is another key topic. Also:

• substitute vs. substituter => this (I think)

See annotations with the substitute tag

binary caches tie in with substitutes somehow; get to the bottom of it. See annotations with the substitute tag.

Maybe this?

We can use required tag for html, for a simple check.

I see what he means, but what would you call this (tag)? "have to remember state"? maybe "have to remember" is close enough

Or maybe order is important / do things in the right order is all we need to describe the problem/need.

new tag?:

• code that is universally relevant/valuable
• non - _-specific logic

It seems like GIFs became more popular with social media. It's almost as if social media reinvented the GIF.

What is the purpose of this paragraph?

What is the main idea of this paragraph?

I think tagging the type of annotation that is being made is one of the best ways for students to be consciously aware of the moves they are making as readers.

Mount Judi, also known as Qardū, is Noah's apobaterion or "Place of Descent", the location where the Ark came to rest after the Great Flood, according to very Early Christian and Islamic tradition. The Quranic tradition is similar to the Judeo-Christian legend.

https://noahsfloodnoahsark.wordpress.com/tag/iran/

I thought this was just awesome. It really highlights the attitude that has developed among some people. Things like food, water, and air are so taken for granted that they have actually been reduced to "inconsequential" status because they are "marginal" in the scope of the over all economy. Like the only thing in the world that is real are dollars.

SciScore for 10.1101/2021.02.12.430998: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written consent was obtained from all individuals and the study was approved by the local ethics committee (14/8/20).<br>IRB: Collection of plasma samples from COVID-19 patients treated at the intensive care unit was approved by the Ethic committee of the University Medicine Göttingen (SeptImmun Study 25/4/19 Ü)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Further, cell lines were routinely tested for contamination by mycoplasma.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of recombinant human monoclonal antibodies against SARS-CoV-2 spike: VH and VL sequences of Regeneron antibodies Casirivimab/REGN10933, Imdevimab/REGN10987 and REGN10989 (Hansen et al., 2020) were cloned in pCMC3-untagged-NCV (SINO Biologics, Cat: CV011) and produced in 293T cells by SINO Biological (Beijing, China).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>REGN10989</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The human IgG1 isotype control antibodies IgG1/κ and IgG1/λ were produced by transfecting FreeStyle 293-F or 293T cells (Fisher Scientific, Schwerte, Germany, Cat. no. R790-07) with the respective plasmids using the protocol provided with the FreeStyle 293 Expression System (Thermo Fisher Scientific, Cat. no. K9000-01).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were stained with the recombinant human IgG1 antibodies in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) for 20 minutes in ice, washed, incubated with an Alexa Fluor 647-labeled mouse monoclonal antibody against the human IgG1-Fc (Biolegend, San Diego, USA, cat #409320) and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, California, USA respectively)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG1-Fc</div><div>suggested: (Sino Biological Cat# 10702-MM01T-H, RRID:AB_2860221)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Finally, culture medium was added that was supplemented with anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700; not added to cells expressing VSV-G).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The upper portion of the membrane was probed with anti-HA tag antibody (mouse, Sigma-Aldrich, H3663) diluted 1:1,000 in 5% skim milk solution, while the lower portion of the membrane was probed with anti-VSV matrix protein antibody (Kerafast, EB0011; loading control) diluted 1:2,500 in 5% skim milk solution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HA</div><div>suggested: (Sigma-Aldrich Cat# H3663, RRID:AB_262051)</div></div><div style="margin-bottom:8px"><div>anti-VSV matrix protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following incubation over night at 4 °C, membranes were washed three times with PBS-T, before being probed with peroxidase-conjugated anti-mouse antibody (Dianova, 115-035-003, 1:5,000) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID:AB_10015289)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 293T (human, kidney; ACC-635, DSMZ), Huh-7 (human, liver; JCRB0403, JCRB; kindly provided by Thomas Pietschmann, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany) and Vero76 cells (African green monkey, kidney; CRL-1586, ATCC; kindly provided by Andrea Maisner, Institute of Virology, Philipps University Marburg, Marburg, Germany) were cultivated in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS, Biochrom), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero76</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Caco-2 (human, intestine; HTB-37, ATCC) and Calu-3 cells (human, lung; HTB-55, ATCC; kindly provided by Stephan Ludwig, Institute of Virology, University of Münster, Germany) were cultivated in minimum essential medium supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (Thermo Fisher</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HTB-37</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 cells (human, lung; CRM-CCL-185, ATCC) were cultivated in DMEM/F-12 medium with Nutrient Mix (Thermo Fisher Scientific) supplemented with 10% FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following experimental set-ups were used: (i) In case of experiments comparing the efficiency cell entry by WT and mutant SARS-2-S, target cells were inoculated with 100 μl/well of the respective pseudotype particles; (ii) For investigation of inhibition of SARS-2-S-driven cell entry by the serine protease inhibitor Camostat mesylate, Calu-3 cells were preincubated for 1 h with medium (50 μl/well) containing either increasing concentrations of Camostat (0.5, 5 or 50 μM; Tocris) or dimethyl sulfoxide (solvent control) before the respective pseudotype particles were added on top; in order to assess the ability of sol-hACE2-Fc, patient sera and monoclonal antibodies to block SARS-2-S-driven cell entry, pseudotype particles were preincubated for 30 min with medium containing different dilutions of either sol-hACE2-Fc (1:20, 1:200, 1:2,000) or patient serum/plasma (serum: 1:50, 1:100, 1:200, 1:400, 1:800; plasma: 1:25, 1:100, 1:400, 1:1600, 1:6400), or with different concentrations of monoclonal antibody (5, 0.5, 0.05, 0.005, 0.0005 μg/ml), before being inoculated onto Vero76 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: KCLB Cat# 30055, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of sol-hACE2-Fc: 293T cells were grown in a T-75 flask and transfected with 20 μg of sol-hACE2-Fc expression plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data normalization was done as follows: (i) In order to assess enhancement of S protein-driven pseudotype entry in BHK-21 cells following directed overexpression of hACE2, transduction was normalized against the assay background (which was determined by using rhabdoviral pseudotypes bearing no viral glycoprotein, set as 1); (ii) To compare efficiency of cell entry driven by the different S protein variants under study, transduction was normalized against SARS-2-S WT (set as 100%); (iii) For experiments investigating inhibitory effects exerted by sol-hACE2-Fc or Camostat Mesylate, patient serum/plasma samples or monoclonal antibodies, transduction was normalized against a reference sample (control-treated cells or pseudotypes, set as 100%).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK-21</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence alignments were performed using the Clustal Omega online tool (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Clustal Omega</div><div>suggested: (Clustal Omega, RRID:SCR_001591)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein models were designed using the YASARA (http://www.yasara.org/index.html) and UCSF Chimera (version 1.14, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco) software packages, and are either based on PDB: 6XDG (Hansen et al., 2020) or on a template generated by modelling the SARS-2-S sequence on a published crystal structure (PDB: 6XR8, (Cai et al., 2020)) with the help of the SWISS-MODEL online tool (https://swissmodel.expasy.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>YASARA</div><div>suggested: (YASARA, RRID:SCR_017591)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data normalization and statistical analysis: Data analysis was performed using Microsoft Excel as part of the Microsoft Office software package (version 2019, Microsoft Corporation) and GraphPad Prism 8 version 8.4.3 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The following limitations of our study need to be considered. We employed pseudotyped particles instead of authentic SARS-CoV-2 and we did not determine whether Y453F affects viral inhibition by T cell responses raised against SARS-CoV-2. Further, we did not investigate whether presence of Y453F in the SARS-CoV-2 S protein increases binding to mink ACE2. Nevertheless, our results suggest that the introduction of SARS-CoV-2 into mink allows the virus to acquire mutations that compromise viral control by the humoral immune response in humans. As a consequence, infection of mink and other animal species should be prevented and it should be continuously monitored whether SARS-CoV-2 amplification in other wild or domestic animals occurs and changes critical biological properties of the virus.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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Results from JetFighter: We did not find any issues relating to colormaps.

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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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[Geisel, 2017] Using social bookmarking tools to bookmark, tag, annotate, and take notes on websites; "Better bookmarking"

• Diigo
• Hypothes.is

Summary: This study provides new information about how amyloid beta (Ab) oligomers (Abo) may contribute to hyperexcitability which is important because Abo and hyperexcitability have been suggested to occur early in the development of Alzheimer's disease (AD). The authors added Abo intracellularly (iAbo) using dialysis from a patch pipette. Their data suggest iAbo led to increased synaptic excitation mediated presynaptically by retrograde signalling of nitric oxide (NO). Furthermore, they present data suggesting that there is spread of this increase in excitation to neighboring neurons.

Major Comments:

1) The nature of the described effects of intracellular iAbo are quite unexpected, occurring within a minute of obtaining intracellular recording configuration, which contrasts with at least on previous study. While some controls for intracellular application of oligomers are provided, with reverse iAbo failing to reproduce the effect (Fig 2S1) and the effect being blocked by the antibody A11 (Fig 2S2), further controls are necessary to explain this rapid effect, which seems faster than that for the diffusion of the fluorescent tag into the cell (Fig 1S1). Note that Pusch and Neher (Pflug Arch 1988) determined diffusion time for different substances. That paper or others should be cited, and then some estimation of equilibrium time based on diffusibility of ab oligomers should be provided. Equations 17 and 18 in that paper provide some estimates based on molecular weight or diffusion coefficient. One point in Pusch and Neher is there is extreme variability between access times across cells and that it depends on access resistance, of course. Finally, the Pusch and Neher calculations were for small spherical cells - diffusion into spatially extended cells with long dendrites where the synapses are will take even longer. This is especially critical, as one of the major papers of precedent for this work is that of Ripoli, et al. 2014 (cited in the manuscript) in which the authors of that work examined effects of patch applied Ab42 over the course of 20 minutes, with internal controls showing differences between initial responses, right after break in, and 20 minutes later when the oligomer and/or monomers will have had a chance to equilibrate with the intracellular contents. It is not clear how such a rapid effect as indicated in the figures could be achieved by such a large molecule as Ab. The data suggest a time to effect of seconds to minutes, and the peak effect occurs before the fluorescence peaks, which seems hard to explain.

2) The data need reorganization in terms of their results using h-iAbo or iAbo. There needs to be a clear demonstration of why both were used if the results are generalized with both (or not) and if they can actually use both interchangeably.

3) The authors need to clearly indicate whether the experiments were done in culture or in slices. The authors need to provide a rationale on why specific experiments were done in culture and others in slices.

4) There are aspects of the observed phenomenon that have not been taken adequately into account. For example, the authors have not investigated the effects of application of oligomeric beta-amyloid to either the extracellular space or the presynaptic neurons, two other compartments of the synapse.

5) Aspects of the data raise questions: 1) Western blots appear to have multiple bands 2) evidence that the fluorescent probe accurately measures NO. 3) The bursts of activity are not quantified. What was defined as a burst? What was the burst frequency and did it change over the recording period? 4)The external solution for cultures contain 5.4 mM K+ which is quite high, and can induce hyperexcitability. Similarly, the use of 100uM AMPA and GABA seem very high. Justifying these high concentrations is important. They should lead to hyperexcitability and toxicity (AMPA) over time. Another point of concern is that the concentration of K+ for the slice work is 3 mM, much different than cultures. There are also differences in Mg2+ and Ca2+, making data hard to compare in the two preparations. 5) sample sizes are unclear 6) Intracellular Ab produces increases in both EPSCs and IPSCs. However, in Fig 3, the IPSC measures using a charge transfer quantification, did not show a significant change in response to iAbo, in contrast to EPSCs. 7) With regard to the inhibition, In the schematic on Fig. 10, I find this incomplete and slightly inaccurate since it shows one terminal releasing both glutamate and GABA with NO increasing both. While this is obviously an oversimplification, it's slightly inaccurate since NO was not directly shown to increase sIPSCs. Were NOS blockers able to disrupt the increase in sIPSCs? Moreover, there are many papers that have shown that PKC can also phosphorylate GABA receptors and increase their conductance. What could be the reason that this was not involved here? This needs to be discussed.

6) How this work relates to other studies is necessary. For example, how this study is related to others about Ab exposure is lacking. Also, regarding hyperexcitability, many possible causes exist. These should be summarized in the introduction and the authors should comment how their results fit with these studies. Regarding PKC and NO, PKC and NO have several known actions throughout the brain and body. How do the effects the authors have identified relate to all these other effects? For example, if PKC is activated by another mechanism, would it occlude effects of Ab? What are the changes in PKC and NO in AD? Regarding the ability of the data to address AD, a major issue is whether the results are relevant to AD or represent interesting pharmacological data about what Ab can potentially do in some of its forms in normal tissue.

Reviewer #2 opted to reveal their name to the authors in the decision letter after review.

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

Learn more at Review Commons

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Reviewer #4

Evidence, reproducibility and clarity

We have reviewed "A specific regulator of neuronal V-ATPase in Drosophila melanogaster." by Dulac et al. The authors have identified VhaAC45L as a regulator of neuronal V-ATPase in Drosophila melanogaster. The authors have utilized multiple techniques to determine the localization of VhaAC45L in neurons and specifically in the synapse. The use of multiple approaches including determining RNA levels in different regions of the fly, and using CRISPR-Cas9 technique to insert V5 tag, makes a very convincing argument about the synapse-specific expression of VhaAC45L.

The combined use of co-immunoprecipitation technique and LC/MS to show that VhaAC45L co-precipitated with V-ATPase complex subunits is convincing that VhaAC45L is a subunit of V-ATPase. To determine the role of VhaAC45L in acidification of synaptic vesicles the authors have utilized pHluorins in combination with multiple RNAi lines. The authors have used a well-designed experiment to prove that VhaAC45L regulates acidification of the synaptic vesicles. Further, larval locomotion and quantal size determination using VhaAC45LRNAi which is known to be altered due to pH gradient of synaptic vesicles shows the functional role of VhaAC45L in synaptic vesicle acidification.

Minor comments:

1. For all graphs, please remove gridlines to make data points more visible.
2. Line 120-123: Authors indicate the VhaAC45LRNAi induced lethal phenotype when expressed in glutamatergic and cholinergic drivers but the figure is missing. Please indicate as "data not shown" if not included in Figure.
3. A diagram summarizing the role of VhaAC45L in V-ATPase enzymatic complex and specific role is recommended.

Significance

V-ATPase play a crucial role at the synapse by being responsible for acidification of the synaptic vesicles and identification of a synaptic vesicle specific regulator of V-APTase is important to understand the complex regulation of synapse function. The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.

Referees cross commenting

The summary of Reviewer#2 looks good!

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

Learn more at Review Commons

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Reply to the reviewers

We would like to thank the editors and the four reviewers for their careful consideration of our manuscript. We are very grateful for their positive appreciation of our work and we believe that their suggestions, which have been included in the preliminary revised version of the manuscript whenever possible, have greatly improved the quality of the paper and have helped us deepen our understanding of the results.

We were happy to note that all the reviewers found value in our work, as stated in their general comments: “This is certainly a useful contribution to our understanding of neuronal V-ATPase functions in vivo” (…)” (Reviewer 1) – “Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase. (…) The experiments are very well performed, the data presented very convincing and the paper is well written.” (Reviewer 2) – “The discovery of a neuronal specific regulator of the V-ATPase is very interesting (…) The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.” (Reviewer 3) – “The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.” (Reviewer 4).

In their remarks, the reviewers suggested additional experiments that could be done to improve our understanding of the role of this new V-ATPase regulator, as well as several minor issues. We have addressed all their comments in our answers below, in which the full text of the reviews is included in blue type, and the responses in black. The line numbers refer to the revised version of the manuscript.

Reviewer #1

Dulac et al. present a first in vivo characterization of the 'accessory' v-ATPase subunit vhaAC45L in Drosophila. The key findings are localization and association of the protein with v-ATPase complexes at synapses and a functional requirement based on lethality and reduced synaptic function. This is certainly a useful contribution to our understanding of neuronal v-ATPase functions in vivo. The main weakness of the study is a lack of depth. The study focuses on localization, co-IP of associated proteins, an analysis of acidification and reduced synaptic function in fly larvae, thus providing a baseline for mechanistic study. However, the mechanism of vhaAC45L is not addressed in this short report. How does is vhaAC45L function different from its homolog vhaAC45? Is it required for v-ATPase assembly? Is it required to localize the full v-ATPase complex (or just V0) to the synapse? Is the defect really due to partial loading of synaptic vesicles or does loss of vhaAC45L also affect endosomal and lysosomal function at synapses? The work as is certainly represents a publishable contribution without answering any of these questions - more as an invite for the community to study the role of vhaAC45L; however, I feel this is a bit of a missed opportunity to put the function of a new potential regulator of specific synaptic v-ATPase functions in the context of the most basic functions obvious in this field.

My main concerns are:

1. clearly, vhaAC45L is required for SOME function of v-ATPase in neurons - but it remains entirely unclear which one. It is not even clear what compartments are affected. Reduced quantal size of single vesicle exocytosis events can be a direct or indirect consequence of problems in SV biogenesis and recycling.

Is exo- /endocytosis unaffected? (FM1-43 uptake!).

We agree that alterations in the synaptic vesicle release/recycling cycle could indeed contribute to the locomotion defect, in addition to the acidification impairment observed in VhaAC45L knockdown larvae. As suggested by the reviewer, we plan to carry out FM-dye assays to measure endocytosis and exocytosis at the neuromuscular junction of control versus VhaAC45L-KD animals. If successful, a new figure will be added to the final version of the paper.

What compartments are affected? (markers for synaptic vesicles versus lysosomal compartments!).

Finding out whether VhaAC45L is specifically involved in the acidification of synaptic vesicles, or if it also plays a similar role in other synaptic organelles, in particular lysosomes, would be very interesting indeed. However, we found that it was technically difficult to address this issue in the Drosophila nervous system. A good way would be to check whether the lysosomal pH is affected by VhaAC45L knockdown, as it is the case for synaptic vesicles.

Unfortunately, because lysosomes are not abundant in neurons, lysosome-specific pH-sensitive probes such as Lysotracker do not yield detectable signals at Drosophila larval synapses. So, whether VhaAC45L is specific for synaptic vesicles or involved in the regulation of V-ATPase activity in all neuronal compartments reminas an open question for now.

1. molecular function: is vhaAC45L required for v-ATPase assembly? (IP/Pull-downs of v- ATPase complexes in the presence or absence of vhaAC45L with other subunits!).

In accordance with the reviewer, we are also very much eager to learn more about the precise molecular function of VhaAC45L, and in particular whether it is required or not for assembly of the V-ATPase complex. Pull-downs of V-ATPase proteins in controls versus VhaAC45L-KD could be used to address this question, but this would require a large quantity of antibodies directed against subunits of the V0 and V1 domains, respectively. Unfortunately, there are no such antibodies commercially available against Drosophila V-ATPase proteins. We have tried several antibodies that recognize V-ATPase subunits from other species and were predicted to react against Drosophila homologs, but with no success. The only V-ATPase antibodies currently at our disposal were samples generously sent to us by other laboratories in insufficient quantities for carrying out such experiments. To our regret, therefore, we were not able to answer this question until now because of the lack of appropriate tools.

1. vha100 was proposed in Drosophila to function on synaptic vesicles and the lysosomal pathway, but, if I remember correctly, here quantal size was normal. I am missing a comparison between the two.

We thank the reviewer for this comment. A comparison with previously published results on subunit Vha100-1 has now been added (lines 458-469) in the discussion related to this topic in the revised manuscript.

1. The V5 knock-in is used both as a mutant as well as a tool to analyze protein localization. This is likely okay, but a little concern of course has to be that by creating a mutant protein through stop codon deletion its subcellular localization, turnover, etc. are not normal. Similarly, anti-V5 co-IPs will isolate proteins bound to the mutant variant of vhaAC45L. Minimally, IPs or pull- downs using other members of the V0 complex should be done to understand the role of vhaAC45L in direct comparison with vhaAC45 on complex assembly and possibly targeting to the synapse (or ideally targeting to specific compartments).

It is indeed a legitimate concern to question the physiological relevance of results obtained by studying V5-tagged VhaAC45L. However, the V5 tag is very small (14 amino acids) and we fused it in place of the stop codon to keep intact the whole sequence of the protein. In addition, we found that the V5 knock-in flies are viable and fertile as homozygous. Given that the null mutants, as well as strong RNAi knockdowns, are lethal at early developmental stage, this suggests that the V5 knock-in has limited negative effects, if any, on VhaAC45L function. This led us to believe that at least a good portion of the V5-tagged protein might be targeted to the right subcellular compartment, and associate to its physiological partners.

Significance:

There is significance to the reporting of an accessory v-ATPase subunit required for SOME function of the v-ATPase in neurons. There is some lack of significance in the absence of basic mechanistic insight as to what vhaAC45L does to the v-ATPase in neurons.

We agree that we did not elucidate here the precise molecular mechanisms by which VhaAC45L contributes to synaptic vesicle acidification. It is rather an initial description of a novel neuronal protein that appears to be essential for proper synaptic functioning, and we provide consistent evidence that its function requires specific interaction with the V-ATPase complex, and in particular with three subunits that reproducibly co-immunoprecipitated with VhaAC45L (namely Vha1C39-1, Vha100-1 and ATP6AP2). Please note that it took many years and many papers before the molecular mechanisms of action of comparable accessory subunits, such as ATP6AP1/AC45 or ATP6AP2, was better understood, and it is still nowadays a matter of investigation. It is therefore very demanding to expect that we describe the exact function of the previously uncharacterized VhaAC45L at all levels in a single first paper.

Reviewer #2

In this study and using Drosophila melanogaster as a model system, Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase called VhaAC45L. They combine genetics, IHC, Mass spec and ephys to unravel the expression pattern and function of this protein. They find that it is required to acidify synaptic vesicles in glutamatergic neurons of the Drosophila larval neuromuscular junction, for appropriate synaptic transmission and for larval locomotion. The experiments are very well performed, the data presented very convincing and the paper is well written. Nonetheless, a few additional pieces of evidence and some level of expanded analysis would strengthen the conclusions and increase the depth of the work.

Major comments:

1. Figure 1F: the while the localization to the presynaptic terminal is convincing, where exactly the protein is localized to is not studied. The imaging in these experiments could use increased resolution and concomitantly colocalization studies with more specific synaptic vesicle markers.

We agree that it would be very good to show this additional result. However, confocal microscopy does not provide sufficient resolution to localize the protein at the membrane of individual synaptic vesicles. Another way would be to see if VhaAC45L immunostaining co- localizes with domains enriched in synaptic vesicle markers, but these organelles are rather ubiquitously distributed in the synaptic boutons at the Drosophila neuromuscular junction. To correctly perform this experiment, we would have to do immuno-electron microscopy, a technique we do not master in our laboratory and that we did not plan to implement for the present work.

1. Figure 3B-G: these experiments should be complemented by a rescue experiment, ideally of the null mutant using a UAS construct and a pan neuronal driver, or - if such animals are viable to the third larval instar stage - a glutamatergic driver. If possible, it would also be good to study the NMJ phenotype of the null mutant rescued to viability using a neuronal driver that does not express in motor neurons (e.g. Chat-G4).

Although a rescue experiment could potentially add a further evidence that Vha45ACL deficiency is responsible for the synaptic vesicle acidification defect described in Figure 3, we don’t think that it is a requisite here because we obtained similar results by knocking down the gene using two different RNAis. As described in the manuscript, the pan-neuronal expression of Vha45ACL could rescue the embryonic lethality of the null mutant, so it would be theoretically possible to check the acidity level of synaptic vesicles at the neuromuscular junction of the recued larvae. However, this would involve making rather complex genetic constructions to express VMAT-pHluorin in motor neurons in rescued mutant background. In addition, the conclusions we could draw from such experiment would be limited by the lack of comparison. Indeed, in Figure 3 the defect was observed in knockdown context, and the same experiment could not be performed in knockout larvae due to the early lethality. If we could measure the acidity level of rescued null mutants, we would not have any comparison point besides the knockdown experiments. As knockout and knockdown are not likely to produce identical phenotype (especially in terms of magnitude of effect), the ideal would be to compare the rescued phenotype to the null mutant expressing VhaAC45L in all neurons except motoneurons, as suggested by the reviewer. However, such genotype would certainly not be viable, since we observed that expression of VhaAC45L RNAis with a stronger motoneurons driver (D42-Gal4) was sufficient to induce lethality at early developmental stage.

1. Figure 5: the authors focus on quantal size which measures the postsynaptic response to spontaneous release from the presynaptic terminal. However, it is unclear how this directly relates to the locomotor deficit beyond signaling potential deficits in vesicle loading or fusion. It would be more convincing to also study evoked release, and expand the analysis of presynaptic properties (number of events, amplitude, frequency).

We fully agree with this comment shared by Reviewers 2 and 3 related to the electrophysiology experiments. Note that these experiments have been carried out in collaboration with another laboratory located in another city. The Covid-19 situation during the past year has prevented, and is still complicating, movements between labs, preventing us from going further with the electrophysiology analyses of VhaAC45L KD. If the situation in the near future allows it, we would very much like to add a more extensive electrophysiological analysis, including in particular the study of evoked release. In the revised manuscript, we have nevertheless completed Figure 5 by adding representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae, as well as the results of new analyses showing lack of effects the KD on the mean EPSP frequency.

1. General: showing some level of genetic interaction with V-ATPase subunits in at least some of the assays would be welcome.

We are definitely in accordance with the reviewer on that point, but we think that this would involve a lot of work and be beyond the scope of the present initial description. Here we show by proteomic analyses that at least 12 proteins co-precipitate and so potentially interact with VhaAC45L, three of them being previously identified constitutive or accessory V-ATPase subunits. In our opinion, studying the interactions between VhaAC45L and these proteins through genetic and molecular studies will be the subject of future works. As stated by Reviewer 2 in the Referees cross commenting below: “further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time”. We fully agree with this statement.

Minor comments:

Some of the images, especially those in Figure 3, should be larger for ease of visualization.

As requested, the images of Figure 3 have been enlarged.

Significance

The discovery of a neuronal specific regulator of the V-ATPase is very interesting. To my knowledge it is the first description of a neuronal specific V-ATPase related protein since the description of Vha100-1 by Hiesinger and colleagues in 2005. The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.

We are grateful to the reviewer for his very positive assessment of our work.

I note that I do not have in depth expertise in electrophysiology, although I am sufficiently familiar with basic NMJ physiology experiments to render the opinions stated above.

Reviewer #3

In this study, Dulac and colleagues investigated roles of VhaAC45-like gene, which codes one of the V-ATPase accessory proteins in Drosophila, in synaptic transmission. First, they demonstrated that VhaZC45L transcripts are expressed selectively in neurons and that the gene products are addressed to synaptic areas. Second, they showed that VhaAC45L is co- immunoprecipitated with some subunits of V-ATPases, which is consistent with bio-informatics predictions. They further demonstrated that VhaAC45L-knockdown (KD) resulted in defects in synaptic vesicle acidification as well as a reduction in quantal size of glutamate, indicating that VhaAC45L play a key role in regulating neurotransmitter release by modulating the driving force for transmitter uptake. Last, not least, they demonstrated that VhaAC45L-KD in motoneurons attenuated larvae locomotor performance, indicating its physiological relevance. Overall, this study is rigorously executed and nicely presented, and adds one more component of the V- ATPase that is responsible for neurotransmitter uptake into synaptic vesicles. However, since this study simply confirmed an established notion from other species such as yeast and mammals that AC45 is one of the accessory proteins of the V-ATPase complex, a conceptual novelty beyond the previous knowledge is relatively poor in its present form. Thus, this reviewer would suggest several issues as following to improve the comprehensiveness as well as novelty of the current manuscript.

1. The reason why the authors focused on VhaAC45-'like' is somewhat obscure, and therefore should be explained. How different VhaAC45 and VhaAC45L are in terms of amino acid sequences, tissue distributions, and KO phenotypes. It seems more comprehensive if the authors provide some experimental evidence on VhaAC45; e.g. whether it is also expressed in neurons or not (Fig. 1), and, if VhaAC45 is neuronal, whether it can rescue the phenotypes of VhaAC45L- KD to certain degree (Figs 4 & 5).

Following the reviewer’s request, we have added a sequence alignment of VhaAC45 and VhaAC45L, as well as a graph showing tissue distributions of both genes in Supplementary Figure 1 of the revised manuscript. To our knowledge, there is no published functional study of VhaAC45 in Drosophila, so we can only make assumptions derived from studies on predicted homologs in evolutionarily distant species. For that reason, it is difficult to compare VhaAC45 to VhaAC45L, as it would first require an entire new study of VhaAC45 function in flies. Since our interest is to study neuronal physiology, we focused on VhaAC45L because compelling evidence indicates that this subunit is specific to the nervous system, as described in our manuscript, rather than on VhaAC45 which seems to be expressed in all tissues. In addition, homologs of VhaAC45L have never been functionally characterized to date in any species, making it very interesting to study this new protein in a genetically tractable organism.

1. What is the mechanism of Ac45 in regulating V-ATPase activity? In mammals, it has been suggested that Ac45 is essential for proper sorting of the V-ATPase to the destined organelles (e.g. Jansen et al., Mol. Biol. Cell., 2010; Jansen et al., BBA, 2008). In this context, it should be examined whether VhaAC45L-KD would affect the synaptic localization of other V-ATPase subunits.

We thank the reviewer for pointing out these very interesting references. We have indeed tried to determine the relative abundance of two other V-ATPase subunits at the larval neuromuscular junction in control and VhaAC45L knockdown contexts. However, because the tested subunits are not specific to neurons, and are expressed at relatively low levels in synapses, it was not possible for us to properly separate the synaptic signal from the background immunostaining in surrounding muscles. This unfortunately prevented us from performing an accurate and reliable quantification.

1. Given that a rodent brain SV contains a few copies of the V-ATPase on average (Takamori et al., 2006, and some newer papers by others), it is interesting that >80% reduction of Ac45 showed moderate effects on quantal size. If SVs under study also contains 1 or 2 V-ATPase per SV, there must be some SVs lacking VhAC45L upon KD. In this context, it is interesting to see how VhaAC-KD (RNAi1~3) affect the frequencies of minis.

The reviewer’s valuable comment prompted us to undertake new analyses on our electrophysiological recordings. We have now added in Figure 5E graphs showing the mean EPSP frequency for larvae expressing VhAC45L RNAi1 and RNAi2, which are the ones that were used in the quantal analysis. Both of these RNAi apparently decreased the frequency compared to controls, but this difference was not statistically significant. As detailed in the Discussion (line 458-469), this may suggest that VhaAC45L does not influence the abundance of the V-ATPase complex at nerve terminals, but rather its efficiency.

1. In general, decrease in mini amplitudes is accounted for by changes in postsynaptic sensitivity for neurotransmitters. Although acidification deficits would support that decrease in quantal size is due to the decrease in the driving force for glutamate uptake, it should be examined whether the postsynaptic receptor fields are not affected by VhaAC45L-KD by recording postsynaptic response upon application of non-saturable concentrations of glutamate.

Testing for potential postsynaptic receptor field alteration by glutamate application would be an interesting experiment indeed, but, as we believe, not a critical control for the present manuscript. Because we expressed RNAis presynaptically, any modification in the postsynaptic receptor field would have to be an indirect consequence of VhaAC45L downregulation in motoneurons, and so, likely to be related to the synaptic vesicle acidification defect. It would not change, therefore, our conclusion that VhaAC45L deficiency in motoneurons induces a decrease in quantal size. Because electrophysiology experiments were carried out in collaboration with another laboratory located in another city, the current sanitary context has so far prevented us from performing this test (please refer to our answer to comment 3 of Reviewer 2 for more details).

1. Related to 4, it is also interesting to see if evoked responses are also attenuated as a result of VhaAC45L-KD, which is more physiologically relevant for locomotor activity phenotype than minis.

We also agree with this comment, shared by Reviewer 2, to which we already responded above in our answer to comment 3 of Reviewer 2.

Minor points:

1. Quantal size of glutamate is not affected by reduced expression of DVGLUT (Daniels et al., Neuron, 2006), which highly contrasts with VhaAC45L, expression of which defines quantal size. Distinct regulation of quantal size by the transporter and the V-ATPase subunit should be discussed.

As suggested by the reviewer, a discussion of this point has been added (lines 458-469). and Daniels et al. 2006 is now cited in the revised manuscript.

1. For electrophysiological experiments, respective sample traces should be shown in Figure 5.

Quantal size is not directly visible in sample traces, so we added instead representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae in the new Figure 5C.

1. <![endif]>Only RNAi1 and RNAi2 lines were examined for SV pH estimation and mini analysis. The results from RNAi3 should be presented, or at least mentioned in the text.

These experiments were performed using two different RNAi constructs to ensure that similar effects were observed and to exclude the possibility of potential off-targets. Knocking down VhaAC45L in neurons with RNAi1 and 2 was lethal at pupal stages, suggesting that they give similar levels of inactivation. RNAi3 systematically induced lighter phenotypes, producing viable adults, which led us to believe it had a lower efficiency. Because the results on synaptic vesicle acidification and electrophysiology were very consistent with RNAi1 and RNAi2, we considered that it was not necessary to repeat the experiment with RNAi3.

Significance

As mentioned above, as it stands, the authors merely confirmed the pre-existing bioinformatic knowledge on one of the AC45 homologues in Drosophila. The audience of The EMBO Journal might be interested in how different/similar VhaAC45 and VhaAC45-like are, and their functional relevance. In particular, is VhaAC45 also mandatory for the V-ATPase functioning in neurons? Adding some basic information of VhaAC45, e.g. tissue distribution, KO phenotypes, and ability to rescue the VhaAC45-like-KD phenotypes, will certainly improve the comprehensiveness of this study, and capture audience's attention.

As mentioned in our response to point 1 of the reviewer above, we have added more data comparing the structure and distribution of VhaAC45 and VhaAC45L in the revised manuscript. VhaAC45 appears to be ubiquitously expressed whereas VhaAC45L is neuron-specific.

Comparing VhaAC45 to VhaAC45L would require a completely new study of VhaAC45 function, because it has never been done before in Drosophila to our knowledge. This would require repeating all the experiments with this other gene, probably involving two more years of work, and would make for a much longer and very different manuscript. It is understandable that this cannot be envisaged. Because homologs of VhaAC45L have never been functionally characterized to date in any species, we considered that it was worth studying this new protein on its own.

Reviewer #4

We have reviewed "A specific regulator of neuronal V-ATPase in Drosophila melanogaster." by Dulac et al. The authors have identified VhaAC45L as a regulator of neuronal V-ATPase in Drosophila melanogaster. The authors have utilized multiple techniques to determine the localization of VhaAC45L in neurons and specifically in the synapse. The use of multiple approaches including determining RNA levels in different regions of the fly, and using CRISPR- Cas9 technique to insert V5 tag, makes a very convincing argument about the synapse-specific expression of VhaAC45L.

The combined use of co-immunoprecipitation technique and LC/MS to show that VhaAC45L co- precipitated with V-ATPase complex subunits is convincing that VhaAC45L is a subunit of V- ATPase. To determine the role of VhaAC45L in acidification of synaptic vesicles the authors have utilized pHluorins in combination with multiple RNAi lines. The authors have used a well- designed experiment to prove that VhaAC45L regulates acidification of the synaptic vesicles.

Further, larval locomotion and quantal size determination using VhaAC45LRNAi which is known to be altered due to pH gradient of synaptic vesicles shows the functional role of VhaAC45L in synaptic vesicle acidification.

Minor comments:

1. For all graphs, please remove gridlines to make data points more visible.

We found that gridlines can be helpful for the readers to assess approximate values on the graphs. So, we have not removed them but rather changed the colour to a light grey so it does not affect any more visibility. We have also placed the points over the error bars in all the graphs, so they become more apparent.

1. Line 120-123: Authors indicate the VhaAC45LRNAi induced lethal phenotype when expressed in glutamatergic and cholinergic drivers but the figure is missing. Please indicate as "data not shown" if not included in Figure.

This mention has been added in the manuscript (line 125).

1. A diagram summarizing the role of VhaAC45L in V-ATPase enzymatic complex and specific role is recommended.

We believe that it is too early in this first report to draw an accurate diagram summarizing the role of this new protein in the V-ATPase complex.

Significance

V-ATPase play a crucial role at the synapse by being responsible for acidification of the synaptic vesicles and identification of a synaptic vesicle specific regulator of V-ATPase is important to understand the complex regulation of synapse function. The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.

We thank the reviewer for his very positive appreciation of our work.

Referees cross commenting

(Written by Reviewer 2)

There seems to be overall consensus among the reviewers on 3 issues:

1. A somewhat more precise understanding of the role of vhaAC45L in the synaptic vesicle cycle through better localization studies and some classic assays (like FM dye uptake).

—See our answers to comments 1 of Reviewer 1 and Reviewer 2.

1. A little more characterization of the transmission defects (e.g. studying evoked responses) would be welcome.

—See our answers comment 3 of Reviewer 2.

1. Ascertaining the validity of the alleles with rescue experiments, perhaps in the V5 mutant background to allow localization analysis in a rescued background.

—See our answers to comment 2 of Reviewer 2.

I think further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time.

We fully agree with this statement.

The minor issues are easy to address

We have addressed all of them in the preliminary revised version of the manuscript.

hiding __?

reviews/ratings because only those already inclined to like it (or who have been swayed by the already positive reviews) will bother buying it and (therefore) bother reviewing it, hence amplifying the positive ratings

Will the crawlers lower the relevance of an H6 tag on my page compared to an H1 tag on someone else's page?

Author uses it in the context of a label, something that can not be easily removed. Used to identify someone

苹果准备最早明年发布它的高端 VR 设备，这款计划中的产品售价将会高达 3000 美元。代号为 N301 的 VR 设备使用了两个 8K 显示屏，采用 M1 芯片的继任者，能展示丰富的 3D 图形。苹果将会使用眼球跟踪技术以较低的保真度渲染非用户注视的目标。苹果正在测试的一个版本使用了 10 多个摄像头，从跟踪手运动到提供周围空间的即时动态，可用于混合和增强现实体验，不限于浸入式的 VR。

Example question. Note the "question" tag.

在这里，墙上和桌子上的每一张纸本质上都是Lua编程语言的一个片段。有些纸上印着工作代码。Dynamicland 实际上可以对该代码执行 OCR 并实时运行它，但是通常，它在现实世界中象征性地使用这些代码，并在服务器端存储了自己的数字部分。然而，当一个对象发生变化时，该系统可以在打印出来的纸张上突出显示改变的线条，并能够标记与更新后的数字文件不同步的代码表。

soup = BeautifulSoup('<b class="boldest">Extremely bold</b>', 'html.parser')
tag = soup.b
tag.string
# 'Extremely bold'
type(tag.string)
# <class 'bs4.element.NavigableString'>

What is this an example of? self-referencing? self-presentation? duality?

tags garner traction. When doing makeup reviews, it is wise to use tags such as "review", so people can search the tags in hopes to find a review of a product they are interested in but want outsider opinions on prior to purchasing

ex: https://www.instagram.com/skin.withlove/

^ specifically in posts where she holds makeup/skincare items to her face, the comments has the tag "review" in it. For example, the post of her holding the @nyxcosmetics 'Bare With Me Tinted Skin Veil BB Cream' ⠀

DIZZYING

wtf. you do NOT tag names like this unless there are 2 ppl with the same name or Mr&Ms -> X Family

why so much useless history and naming?

ethical appeal is being used in that the tone of voice and narrative (one which offers constructive criticism and guidance) aids in strengthening this author's credibility. Considering reddit often invites trolls, hypocrites, racist/sexist ideologies etc- as it is clear that this author is someone truly trying to help in answering the main question of this reddit tag: is there an afterlife/proof of it? The author's ethos effected responses in that people were able to consider the questions the author brought forth and formulate their own answers, and- trusting the author due to his tone of voice which makes him credible- post them in a separate thread for the author to see.

How would you even describe this comment?

"just doing my job"? but he is (I assume) answering to be nice not because it's his job

"I won't take it personally"? vote my answer up or down, whichever you please

impartial, dispassionate, and objective, perhaps? "just the facts, ma'am"

---

Separately, what is the "Please don't thank me!" for? Is it that politeness? False modesty? Genuine modesty? Or is it rude? Why not allow someone to thank you??

I love del.icio.us bookmarks, that I used to catalog my web surfings, classify and share them. Upon its demise I went for Keep with good Web & Android client but without easy public sharing. Could hypothes.is be used as I did use del.icio.us? Edit2021: Looking at h. again after two years plus.

Or consider what Hatch had to say on the subject of rail bridges, which number 41 on the disused section of the line:

"The section of track running from Port Hawkesbury to Sydney has been out of service since 2014 and no detailed bridge capacity rating information is available...[I]nspections from 2015 indicate substantial corrosion of principal structural members in a number of steel bridges and general deterioration of concrete on all of the structures. In order to establish exactly what work is required for each structure, it would first be necessary to carry out detailed bridge inspections followed by corroded ratings of all bridges. This is beyond the scope of the current report."

They budget $14 million for structural work on the Grand Narrows swing bridge intended to allow it to operate to Class 1 specifications (with trains traveling at 10MPH). They suggest pulling the swing span open and closed using tug boats, but warn this is “not advisable for regular use given the higher risk of damage.”

Basically, Hatch had a dilemma: it is the job of a consultant to give its client (in this case the Port of Sydney) what it wants, and what the Port wanted was a palatable price tag for refurbishing the rail line.

But consultants have to retain some shred of credibility, so Hatch gave the Port its palatable figure, but added so many caveats that -- as I say in the article -- were the actual cost to be quadruple the estimate, it was covered .

It is interesting to see the tag and the definition of it.

I think this part explains the domino effect. A small proposition might lead to such a big change. There must be a lot of works to do.

I want to write about "The Uberable Lighness of Meaning" and got this by searching for

open market operations destroy price discovery

https://hyp.is/ZaLKPF_WEeuE_T_F-wXBMw/dimartinobooth.com/tag/danielle-dimartino-booth/page/4/

destruction of price discovery

Trabajo Superficie Activa

Why note-taking is so valuable.

• for : TrailHub4Hypothesis

yer it ..

Reviewer #2:

This manuscript interrogates function of Ihog and Boi adhesion molecules in cytoneme-based transport of the Hedgehog morphogen in Drosophila. The cell biology of how cytonemes are regulated to deliver morphogen signals is not yet well understood, so the work addresses an important topic that will be of interest to a broad audience. However, much of the study refines previous work from the same group to provide only a modest advance in understanding of how Ihog impacts cytoneme behavior.

The authors use genetic strategies in Drosophila to investigate how Ihog and Boi influence cytoneme dynamics. They find that the two proteins act differently with regard to cytoneme function. Boi effects are not exhaustively analyzed, but a number of genetic experiments are performed to interrogate Ihog. The authors reveal that the extracellular domains of Ihog interact with the glypicans Dally and Dlp to stabilize cytonemes that originate from Ihog over-expressing cells. Knockdown of Ihog does not alter cytoneme dynamics.

The most novel aspect of the study - that Boi functions differently than Ihog in cytonemes - is, unfortunately, not expanded upon. Some experiments lack controls or are presented in a manner that prevents clear interpretation of results.

Key points to be addressed:

Figure 1: Null alleles and RNAi silencing are used interchangeably to reduce Ihog, Boi, Dally and Dlp function in vivo. Results between methods are directly compared. Oftentimes, controls are not included to confirm the level of knockdown following RNAi. If possible use null alleles due to consistency. However, if this is not possible due to experimental reasons, give an explanation and state impact in the discussion.

Ihog levels decrease following loss of Dally or Dlp and Boi levels appear to increase following knockdown of Ihog, Dally, or Dlp. These stability changes have previously been reported. The mechanism is not clear, so should have been investigated here - especially the increased Boi protein level. How does this occur? Is stabilization occurring at the protein level or is gene expression changing? Is this a compensatory upregulation?

Based upon the supplement for Figure 2, it looks like the Ihog truncation mutants show variable stability. Might this be affecting the extent to which they alter Dally or Dlp stability? The western blot data are presented as crops of single bands adjacent to crops of a molecular weight ladder. Blots should be shown as intact images, preferable with all variants compared across a single gel with a loading control. As presented, relative stability/expression levels are impossible to assess.

Figures 3-4: Ihog mutant transgenes are tagged with either HA or RFP. Best to be consistent with tags when mutant function is being directly compared. Given that the HA tag is a small epitope and the RFP is a protein tag, they may differentially alter protein functionality. To be consistent it would be preferable to use the same tags. However, if this is not possible due to experimental reasons, the technical implication can also be mentioned in the discussion.

Figure 5: Investigation of histoblast cytonemes reaching into ttv, botv mutant clones: The ability of cytonemes to invade double mutant clones is altered only under the engineered situation of glypican dysfunction combined with Ihog over-expression. From this, it is concluded that Ihog is acting with glypicans to stabilize cytonemes. This may be the case, but they ability to see it only under an engineered situation of compound mutation plus Ihog over-expression leads this review to question the physiological relevance of the observation. Of similar concern is that the authors state the ability of Ihog over-expressing cell cytonemes to cross small vs. large ttv, botv clones differs. The difference is very difficult to appreciate from the results presented.

Figure 6: The apparent functional difference between Ihog and Boi in the ability to stabilize cytonemes is potentially very interesting, but is not investigated, which limits the advance of the current study.

This made me consider how digital annotation could be a fun way for students to enforce their learning of literary devices when physical class time is not a possibility, or as a way to integrate technology into the classroom in new ways.

AAAAA

WWWWW

SSSSS

DDD

Now there's a touchstone for the ages

A dedicated sponge in the blank countenance of empty space and flat time, I'd say.

10 Principles of Accessibilities:

1. Blindness (covers most of accessibility issues through testing)
2. Images (Alt-Text)
3. Tag hamburger menus
4. Don't place important content out of the way
5. Test for accessibility with real users.
6. Don't disable zoom in mobile interfaces.
7. Accessibility is cheaper when done up front.
8. Be aware of visual bias.
9. Check mobile accessibility separately.
10. Embrace all access attitude.

.h2

Reviewer #3:

The authors present a simple model that explains important outstanding controversies in the field of long-range gene regulation. These controversies include the fact that insulation boundaries tend to be weak; that acute inactivation of CTCF or cohesin (that leads to inactivation of insulation boundaries) leads to only minimal gene expression and that in live cells enhancer-promoter contacts appear not correlated with transcriptional bursting. The model involves a futile cycle of tag addition and removal from promoters, stimulation of more tag addition when tag is already present, and stimulation of tag addition by contacts with distal enhancers. The authors show that such a model explains all the above controversies, and indicate that the controversies are not inconsistent with mechanisms where long-range gene activation is driven by physical contacts with distal regulatory elements.

The authors have explained and explored the properties of the model well. I have only minor comments.

1) An alternative explanation for TAD-specific enhancer action is that an E-P interaction within a TAD (between two convergent CTCF sites), one that is brought about by extruding cohesin, is not equivalent to an interaction that occurs between two loci on either side of a CTCF site and that can be a random collision that is not mediated by extruding cohesin. In other words, two interactions can be of the same frequency but can be of a very different molecular nature. I agree that this model would not explain the results of the experiment where cohesin is acutely removed.

2) In the beginning of the introduction the authors introduce TADS. I recommend that the authors present this in a more nuanced way: compartment domains also appear as boxes along the diagonal, an issue that has led some in the chromosome folding field to be confused. This reviewer believes TADS are those domains that strictly depend on cohesin mediated loop extrusion, whereas compartment domains are not. If the authors agree, perhaps they can rewrite this section?

3) If I understand the model correctly, the nonlinearity arises because of the increased rate of tag addition when tag is already present. The authors then speculate histone modifications can be one such tag. However, there are only so many sites of modification at a promoter. Can the authors analyze how the possible range of tag densities affects performance of the model? Is the range required biologically plausible?

4) Can the authors do more analysis to explore how rapid changes in gene expression may occur (e.g. upon signaling a gene may go up within minutes)? How much more frequent does the E-P interaction need to be for rapid switch to the active promoter state? Can the authors do an analysis where they change the rates of the futile cycle upon some signal: at what time scale does transcription then change (keeping E-P frequency the same)?

free text and tag search

label

Dieses Ziel scheint mir etwas nichtssagend bzw. zu generisch zu sein ohne weitere Ausführungen. (Ich versuche z.B. jeden Tag einen positiven (und nicht negativen) Beitrag zum (Welt-)Geschehen zu machen und denke das gilt für viele andere auch.)

Ebenfalls ist mir nicht klar, in welcher Relation dies alles mit der verlinkten Agenda 2030 steht; da sollte m.E. ein expliziter Bezug im Text gemacht werden.

This is where I've felt the same way and the value of integrating support into the product as opposed to making the users go outside the product (e.g., support portal).

Make it easy to get feedback - whether it's an idea or problem the user using our product runs into.

Reviewer #3:

In the manuscript by Kim et al., show that, beyond its roles of preventing somatic differentiation in the germline of embryos, Zn-finger protein PIE-1 also functions in the adult germline, where it is both SUMOylated as well as interacts with the SUMO conjugating machinery and promotes SUMOylation of protein targets. They identify HDA-1 as a target of PIE-1-induced SUMOylation. Here too, I find the claims interesting, however data is sometimes missing or does not fully support the claims.

Main concerns:

1) A key claim of novelty over previously proposed "glue" functions of SUMO is based on the fact that they find that temporally regulated SUMOylation of a very specific residue in a specific protein is affecting protein activity: The observation that "SUMOylation of HDA-1 only appears to regulate its functions in the adult germline" and not in the embryo together with the finding that "other co-factors such as MEP-1 are SUMOylated more broadly, these findings imply that SUMOylation in the context of these chromatin remodeling complexes, does not merely function as a SUMO-glue (Matunis et al., 2006) but rather has specificity depending on which components of the complex are modified and/or when."

I find this claim poorly supported by the data. In fact, I find that the data supports that multiple SUMOylations contribute to formation of larger complexes: The His-SUMO IP (Fig 2B) brings down far more un-SUMOylated HDA-1 than SUMOylated. This argues for the presence of large complexes with different factors being SUMOylated and many bringing down unmodified HDA-1. The chromatography experiments (Fig 3B-C) also provide hits that are in complex and not direct interactors. Finally, HDA-1 SUMOylation is indicated to regulate MEP-1 interaction with numerous factors (Fig 3D). If all these factors are in one complex, it is hard to imagine how a single SUMO residue would mediate all of these simultaneously. It is quite likely (and not tested) that loss of HDA-1 SUMOylation leads to (partial?) dissociation of a large complex, rather than loss of individual interactions with the SUMO residue of HDA-1. Unlike claimed by the authors, there is no evidence that the "activity" of HDA-1 is regulated by SUMO modification.

2) Based on loss of MEP-1/HDA-1 interaction upon pie-1 RNAi and smo-1 RNAi (Fig 4B), the authors conclude that "SUMOylation of PIE-1 promotes the interaction of HDA-1 with MEP-1 in the adult germline".

The evidence that it is PEI-1 SUMOylation that is affecting MEP-1/HDA-1 interaction is fairly weak. In fact, based on Fig 4A, MEP-1 and HDA-1 interact without expression of PIE-1, and in PIE-1 K68R (sumoylation-deficient), although due to poor labeling of the panel it is not clear whether lane 1 and 4 refer to the WT pie-1 locus without tag or lack of pie-1.

In 4B the HDA-1 band that is present in L4440 but not in pie-1 or smo-1 RNAi is very faint, and in our experience such weak signal is not linear i.e., bands can disappear or appear depending on the exposure. Importantly, according to the data, seemingly unmodified HDA-1 immunoprecipitated with MEP-1 (Fig 4B). This data contradicts the authors' claim that "These findings suggest that in the adult germline only a small fraction of the HDA-1 protein pool, likely only those molecules that are SUMOylated, can be recruited by MEP-1 for the assembly of a functional NURD complex".

Furthermore, the fact that pie-1 and smo-1 depletion eliminate the interaction between HDA-1/MEP1 doesn't mean that the SUMOylation of pie-1 specifically is required for the interaction: perhaps un-SUMOylated pie1, and SUMOylation of something else, are both necessary for the interaction. The authors show that MEP-1 is also SUMOylated (Fig3C). When IP-ing GFP-MEP-1, they precipitate all its modified forms and associated factors. One alternative possibility for why smo-1 RNAi abolishes MEP-1/HDA-1 interaction is that MEP-1 SUMOylation is needed for interaction with HDA-1 (independently of pie-1). (On a side note, why are the authors not including MEP-1 SUMOylation in the model?)

3) On page 13 the authors write: "These findings suggest that SUMOylation of PIE-1 on K68 enhances its ability to activate HDA-1 in the adult germline" and "We have shown that PIE-1 is also expressed in the adult germline where it engages the Krüppel-type zinc finger protein MEP-1 and the SUMO-conjugating machinery and functions to promote the SUMOylation and activation of the type 1 HDAC, HDA-1 (Figure 6)". Activation of HDA-1 is misleading and was never tested. If not performing in vitro assays for HDAC activity, the authors at least need to look at whether pie loss (degron) leads to acetylation of genomic HDA-1 targets and whether it affects HDA-1 (and/or MEP-1) recruitment to these sites. This could be done by ChIP-seq of HDA-1 and H3K9ac in WT and pie-1 degron animals.

Chemie- und insbesondere Pharmaanlagen werden nicht in Tagen oder Wochen, sondern eher in Jahren gebaut. Dass wir überhaupt jetzt schon impfstoffe haben, ist nur deshalb möglich, weil Anlagen bereits vor der Zulassung gebaut wurden, damit ein Impfstoff dann möglichst schnell zur Verfügung stellt. Das bedeutet aber auch, dass jede dieser Anlagen ein Risiko darstellt. Es wäre halt betriebswirtschaftlich schwachsinnig, die Anlagen so auszulegen, dass innerhalb des ersten Monats eine Produktion durchläuft, die für die Welt reicht und danach die Anlage abzureißen.

The SMTP envelope is sent as a series of SMTP protocol units (described in Section 3). It consists of

• an originator address (to which error reports should be directed),

MAIL FROM that refers to the originator (a.k.a., reverse path, backward-pointing address) of the request

• one or more recipient addresses,

Multiple RCPT TO for each "to:" rfc822 message header in the mail data (see annotation)

• and optional protocol extension material.

DATA (see below)

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(See also envelope-vs-mail tags.)

This is NOT an H1 tag

DOI: 10.1016/j.celrep.2020.108538

Resource: (Proteintech Cat# 10000-0-AP, RRID:AB_11042316)

Curator: @Naa003

SciCrunch record: RRID:AB_11042316

Curator comments: GST Tag antibody Proteintech Cat# 10000-0-AP

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What is this?

Almost universal education and this is still a large scale problem.

DOI: 10.1016/j.xpro.2020.100231

Resource: (Covance Cat# MMS-101R-1000, RRID:AB_291262)

Curator: @Naa003

SciCrunch record: RRID:AB_291262

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What is this?

Reviewer #1:

My general assessment of this work is that it is full of good ideas and presents a novel and general approach to examine lipid remodeling in cells and perhaps subsequent transport of lipids, mainly to mitochondria, but it lacks the scientific rigor necessary to be fully confident that their conclusions firmly support their claims. Often, insufficient information about the methods are provided and the manuscript is hard to follow critically.

More specific comments:

1) I am surprised that acyl-CoAs are transported into cells. I don't know of any precedent for this. Usually fatty acids are imported into cells and then converted to acyl-CoAs as part of the mechanism of import. Could it be that the acyl--CoAs are hydrolysed before uptake only to be reformed inside the cells? I would suggest feeding the NBD-palmitate plus the lysolipids to the cells as a control to see whether this is the case.

2) In fig 1 as an example they choose a region to blow up. As one can see there is a large variation, even in the blowups of mitochondrial labeling and if one looks at the originals the variation is confirmed. How have they chosen these areas? Furthermore, in figure 1 there is quite a bit of label with MLCL outside of the mitochondria, in particular in regions that they did not choose to blow up. What are these structures? Remodeling of MLCL is thought to take place in mitochondria.

3) They speak of transport of lipids from ER to mitochondria, but in fact the demonstration of this is very weak from what they show in the time course in supp fig 1. I am also disturbed by the difference in patterns of the NBD-PA patterns in a and b. They should be the same, but there are problems, maybe focus? I would say anyway that there is no clear evidence that the NBD PA first appears in the ER then goes to mitos. It could be synthesized in both compartments from their data.

4) The product characterization by TLC is insufficient. There are no standards, no characterization. Would they have seen the free NBD-palm by their methods?

5) When they use mutants and find less "transport" the mitochondrial signal as seen by mitotracker is always more diffuse. This indicates to me that there is another problem.

6) In fig 3 the fluorescent pictures do not correspond to what is seen in the quantification. There is more yellow in e than in h.

7) How did they add cholesterol at 50 or 100 micromolar? It is soluble at less than 1 micromolar in aqueous solution. The cholesterol experiments are puzzling. From what we know about StAR protein it recognizes cholesterol not esters. There is no precedent for cholesterol ester transport into mitochondria. Can they rule out that the esters are transported to the surface of the mitochondria and the NBD-Palm cleaved off and transported into the mitochondria?

8) The MAG and DAG experiments are overinterpreted. It could just be a kinetic problem since the MAG gets converted to DAG before TAG

9) They compare to externally added NBD lipids, but we don't know which ones they used. Are they using short chain NBD phospholipids. I could not find this in their manuscript. If they do not have the same NBD-palm in the sn-2 position then the comparison is meaningless.

10) The excitation and emission spectra of their probes are sometimes overlapping. How did they deal with this? Are they sure that they are not seeing FRET?

Good tag line Play on "get rich quick"

There's a bug in Hypothesis (at least the sidebar client) such that it's possible to post annotations with comments to the the public, but if you want to highlight something and make it similarly public, then it's not possible...

I'm using this tag as a workaround. The annotation comment should be a Markdown-style quote (i.e. set off by an ASCII right-pointing angle bracket / less-than sign).

添加 trace 的 build tag

DOI: 10.1016/j.celrep.2020.108490

Resource: (Abcam Cat# ab1874, RRID:AB_302646)

Curator: @Naa003

SciCrunch record: RRID:AB_302646

Curator comments: Rabbit Anti-VSV-G tag Polyclonal Antibody, Unconjugated Abcam Cat# ab1874

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What is this?

DOI: 10.3390/v12121391

Resource: (MBL International Cat# PM021, RRID:AB_592663)

Curator: @Naa003

SciCrunch record: RRID:AB_592663

Curator comments: S Polyclonal Antibody MBL International Cat# PM021

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What is this?

DOI: 10.1016/j.devcel.2020.10.004

Resource: (IMSR Cat# JAX_013591,RRID:IMSR_JAX:013591)

Curator: @Naa003

SciCrunch record: RRID:IMSR_JAX:013591

Curator comments: FVB-Tg(C3-1-TAg)cJeg/JegJ Mus musculus IMSR Cat# JAX:013591

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What is this?

tag?: crowded (how do we disambiguate, make it not ambiguous?)

tag?: what scope of provided features / recommended happy path is needed?

tag?: what scope of provided features / recommended happy path is needed?

its own _

tool-heavy dependence on build tools / heavy/complex build-time

I absolutely love this concept that he did

给每个想法以 书名 为标签

我也是这么想的:)

DOI: 10.1007/s11120-020-00806-y

Resource: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)

Curator: @Naa003

SciCrunch record: RRID:AB_2556564

Curator comments: V5 Tag Monoclonal Antibody Thermo Fisher Scientific Cat# R960-25

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What is this?

DOI: 10.1111/bph.15060

Resource: (Cell Signaling Technology Cat# 13246, RRID:AB_2798161)

Curator: @Naa003

SciCrunch record: RRID:AB_2798161

Curator comments: T7-Tag (D9E1X) XP® Rabbit mAb antibody Cell Signaling Technology Cat# 13246

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What is this?

testing page note for image

Hi Collin, nice project! So these results would be useful for reliable sources to write articles in a way that will increase shares. Do you think social media could also benefit from these results in order to find and tag misleading articles perhaps?

添加阅读的起始时间和结束时间，一方面是表明是否读完，另一方面则是 measure。

添加 推荐人 也是个好想法，增加更多的 connection，无论是书还是人都有更多的 context

但是需要构建一个 tag system，不光是个不同的 source，甚至还可以通用于非 Roam Research 之外，比如 raindrop

Reviewer #3:

In this manuscript, Naetar et al. investigate the role of LAP2α binding to A-type lamins in the nucleoplasm. LAP2α was already thought to be important for maintaining the nucleoplasmic pool of soluble A-type lamins, because knockout of LAP2α has previously been shown to reduce nucleoplasmic signal from an antibody that recognizes the lamin-A/C amino terminus. However, by directly tagging A-type lamins with fluorescent proteins and by using an alternative antibody to stain them, Naetar et al. find that the presence of LAP2α does not appreciably affect the pool of soluble lamins in the nucleoplasm. Instead, they find that LAP2α affects the assembly state of soluble lamins within the nucleoplasm, preventing formation of higher order A-type lamin structures that impede the mobility of telomeres within the nucleus.

There is a lot to like about this paper. I admire the author's mechanistic approach to studying lamin assembly state. The complementary cell biology/microscopy approaches paired with the biochemical approaches in figure 5 lead to an overall convincing story. And finally, I appreciate the efforts the authors made to "show their work," including their genome editing quality control measures.

Major comments:

1) Although I appreciate the transparency of the authors in demonstrating their workflow and quality control measures (see above), some of the terminology makes the manuscript difficult to read. At times it feels more like reading a lab notebook than reading a manuscript. For example, The manuscript would be easier to understand if cell lines were given descriptive names (eg: LAP2α KO, or mEos3.2-lmna instead of "WT#21") rather than continuing to refer to them by the small guide RNA that was used to generate them. A second example: it is nice to show biological replicate data as in figure 1, but it took me a while to figure out that the second and third columns in panels A and B were biological replicates; I spent some time trying to determine which experimental condition was different. Perhaps one biological replicate could be displayed in the main text and the second could be moved to the supplement, especially considering that it appears that only one of the clones was used for the quantifications shown in the bottom panels.

2) Why was the choice made to disrupt LAP2α at the beginning of exon 4? How large are exons 1 and 2, which are not shown in the schematic in the supplemental figures? What percentage of the LAP2α peptide primary sequence is affected by a frameshift mutation at the start of exon 4? Why was this approach preferable to introducing a frameshift mutation closer to the 5' end of the gene? I am concerned that the "LAP2α KO" cells used in the experiments may have some partially functional truncated LAP2α protein.

3) On page 16, the authors describe a set of experiments that are meant to demonstrate that their failure to see a difference in nucleoplasmic A-type lamins in LAP2α mutants is not due to the fluorescent protein tag used, however, instead of looking at untagged lamins, they elect to look at a cell line that has all lmna alleles tagged. Wouldn't it be better to use the LAP2α KO cells from figure 1 and stain with both the 3A6 antibody and the N18 antibody to determine whether untagged lamins behave the same way as tagged lamins? Perhaps this experiment could be added along with the current data, as it would be nice to compare directly between a cell line with all lmna alleles tagged and a cell line with no lmna alleles tagged.

This experiment would also give the authors a chance to compare morphology and overall fitness of cells with all untagged lmna with cells with all tagged lmna, to determine whether the tagged proteins are fully functional. Even if the tagged protein is fully functional, it would be appropriate to add a brief discussion of the possibility that fluorescent tags do perturb lamin-A/C function. After all, many lamin mutations do not cause obvious phenotypes in tissue culture cells, but defects can still emerge during development and aging in the context of an animal.

4) The authors build a convincing case that binding to A-type lamins by LAP2α influences their ability to assemble. But how do cells leverage this relationship for biological functions? Do cells tune the amount of fully soluble vs. partially assembled A-type lamins in the nucleoplasm in order to control nuclear structure or function in response to certain stimuli? Have the A-type lamins in the nucleoplasm been found to be in a different assembly state in different cell types? As the study is currently written, it presents an interesting molecular mechanism but no biological mechanism.