10,000 Matching Annotations
  1. Last 7 days
  2. www.sciencedirect.com www.sciencedirect.com
    Contrasting population structures of reef-building corals and their algal symbionts inform adaptive potential across the western Pacific
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    sciencedirect.com/science/article/pii/S0960982226005270
  3. www.sciencedirect.com www.sciencedirect.com
    Visual processing is a language-agnostic window into heterogeneity in early reading development
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    sciencedirect.com/science/article/pii/S0960982226005257
  4. www.sciencedirect.com www.sciencedirect.com
    Hyocholic acids shape neonatal immune tolerance and microbiota assembly
    10
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    sciencedirect.com/science/article/pii/S1550413126001543
  5. www.sciencedirect.com www.sciencedirect.com
    Scavenger receptor class F member 2 is an intracellular receptor for hepatitis B virus
    18
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    sciencedirect.com/science/article/pii/S009286742600512X
  6. www.sciencedirect.com www.sciencedirect.com
    Estrogen promotes ethanol-induced CYP2E1 upregulation and Period 2 downregulation, leading to worsened cardiac ferroptosis and dysfunction in female rats
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    sciencedirect.com/science/article/pii/S0006295226004272
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    ARID1A loss drives osimertinib resistance by epigenetically suppressing PTEN and activating the Akt pathway in lung cancer
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    sciencedirect.com/science/article/pii/S0006291X26006728
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    A novel TCF12-MAVS signaling axis in astrocytes promotes vincristine-induced neuropathic pain in male mice
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    sciencedirect.com/science/article/pii/S0889159126005775
  9. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Hormonal staining patterns and clinical outcomes in acromegaly: a 15-year single-center study
    6
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    pmc.ncbi.nlm.nih.gov/articles/PMC13183702/
  10. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Astrocytic Accumulation at the Choroid Plexus Attachment Region
    17
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    pmc.ncbi.nlm.nih.gov/articles/PMC13184079/
  11. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Preferential age-related striatal volume preservation in cervical dystonia
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    pmc.ncbi.nlm.nih.gov/articles/PMC13185566/
  12. onlinelibrary.wiley.com onlinelibrary.wiley.com
    Microbial keystone taxa and metabolic signatures in centenarians regulate intestinal homeostasis during aging
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    onlinelibrary.wiley.com/doi/10.1002/imt2.70134
  13. onlinelibrary.wiley.com onlinelibrary.wiley.com
    Sialylation in Colorectal Cancer Rewires Antitumor Immunity at the Peritoneal Metastatic Site
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    onlinelibrary.wiley.com/doi/10.1002/eji.70204
  14. onlinelibrary.wiley.com onlinelibrary.wiley.com
    Proteomic Signatures of Regeneration: Uncovering Extracellular Matrix-Associated Cues in Goldfish Spinal Cord Repair
    20
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    onlinelibrary.wiley.com/doi/10.1002/cne.70170
  15. chemistry-europe.onlinelibrary.wiley.com chemistry-europe.onlinelibrary.wiley.com
    Design, Synthesis, and Evaluation of Coumarin-Rasagiline Hybrids as Multifunctional Neuroprotective Agents
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    chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cmdc.70307
  16. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Zwitterionic Penicillin‐Derived Sulfone Inhibitor for Combating β‐Lactamase‐Mediated Antibiotic Resistance
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    pmc.ncbi.nlm.nih.gov/articles/PMC13183609/
  17. analyticalsciencejournals.onlinelibrary.wiley.com analyticalsciencejournals.onlinelibrary.wiley.com
    Synergistic Anticancer Effects of Akkermansia muciniphila Combined With 5-Fluorouracil Through BAX/BCL2 Dependent Apoptosis in Colorectal Cancer Cells
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    analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/cbf.70234
  18. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Neurovascular–metabolic dysregulation, metabolic connectomics, and metabolic functional changes in Alzheimer's disease: A preclinical and clinical comparison
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    pmc.ncbi.nlm.nih.gov/articles/PMC13183595/
  19. advanced.onlinelibrary.wiley.com advanced.onlinelibrary.wiley.com
    “Membrane-Guided” Repair Strategy: Precision Delivery of GGT1 Degrader for Targeted Repair and Regeneration of Spinal Cord Neurons
    26
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    advanced.onlinelibrary.wiley.com/doi/10.1002/advs.75554
  20. advanced.onlinelibrary.wiley.com advanced.onlinelibrary.wiley.com
    LC3B Mediated SETDB1-Accounted Alcoholic Steatohepatitis via Lipidation-Dependent LAP and Lipidation-Independent Nuclear Stabilization
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    advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202513189
  21. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Experimental and computational methods for allelic imbalance analysis from single-nucleus RNA-seq data
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    pmc.ncbi.nlm.nih.gov/articles/PMC13185290/
  22. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Q1020R in the spike proteins of MERS-CoV from Arabian camels confers resistance against soluble human DPP4
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    pmc.ncbi.nlm.nih.gov/articles/PMC13185592/
  23. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    NT5DC2 inhibits ferroptosis by stabilizing ACSL3 in bladder cancer
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    pmc.ncbi.nlm.nih.gov/articles/PMC13184144/
  24. legrandcontinent.eu legrandcontinent.eu
    Pourquoi la techno-oligarchie est plus dangereuse que le fascisme historique
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    legrandcontinent.eu/fr/2026/05/19/techno-oligarchie-fascisme/
  25. polymarket.com polymarket.com
    班加罗尔3 ： Ilya Ivashka vs Alex Hernandez 赔率与预测 (May 21, 2026) | Polymarket
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    polymarket.com/zh/sports/atp/atp-ivashka-hernand-2026-05-21
  26. www.biorxiv.org www.biorxiv.org
    Hyperactive STAT1 Promotes T Follicular Helper Type 1 Cell Differentiation to Trigger Autoimmunity
    1
    1. Doctorzhang 21 May 2026

      This study is highly intriguing. For years, there has been ongoing debate in the field regarding the differentiation of Tfh and Th1 cells. Some researchers hold that their differentiation pathways are mutually exclusive, while others propose that they derive from the same progenitor cells. Although this research focuses on the mechanism by which STAT1 mediates autoimmunity, it fundamentally elucidates that STAT1 can endow Tfh cells with Th1-like properties. Furthermore, it confirms that IFN-γ neutralization represents a novel therapeutic strategy for the treatment of numerous autoimmune diseases.

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    biorxiv.org/content/10.64898/2026.03.19.713058v1
  27. Local file Local file
    Untitled document
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  28. callyzer.co callyzer.co
    How to Spot Low-Quality Leads in 10 Seconds
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    callyzer.co/blog/the-ultimate-list-of-cold-calling-statistics-in-india/
  29. polymarket.com polymarket.com
    ITF Kutaisi ： Daniil Ostapenkov vs Saba Purtseladze 赔率与预测 (May 21, 2026) | Polymarket
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    polymarket.com/zh/sports/itf/itf-ostapen-purtsel-2026-05-21
  30. www.facebook.com www.facebook.com
    Austin Typewriter, Ink. | Facebook
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    facebook.com/groups/721704878218903/posts/3096582727397761/
  31. www.medrxiv.org www.medrxiv.org
    A portable molecular laboratory for rapid genotyping in the field: application to sickle cell disease
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    medrxiv.org/content/10.64898/2026.05.05.26352080v1
  32. go.gale.com go.gale.com
    Understanding "Koreanness": Racial Stratification and Colorism in Korea and Implications for Korean Multicultural Education. - Document - Gale In Context: High School
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    go.gale.com/ps/i.do
  33. xupsh.gitbook.io xupsh.gitbook.io
    第六章 稀疏矩阵向量乘法 | FPGA并行编程
    1
    1. Bahair 21 May 2026
      如果行k包含任何非0元素，那么rowPtr[k] 将包含当前行的第一个元素

      例如，rowPtr[2]=4，在values[4]中为2，即原数组第2行第一个数

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    xupsh.gitbook.io/pp4fpgas-cn/zheng-wen/06-sparse-matrix-vector-multiplication
  34. www.biorxiv.org www.biorxiv.org
    The mechanosensitive Ca2+-permeable ion channel PIEZO1 promotes satellite cell function in skeletal muscle regeneration
    1
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    biorxiv.org/lookup/doi/10.1101/2021.03.18.435982
  35. yorushika.com yorushika.com
    ヨルシカ OFFICIAL SITE
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    yorushika.com/lyrics/detail/65/
  36. academic.oup.com academic.oup.com
    Introduction
    2
    1. gmascareno2028 21 May 2026
      Equally, democratic governments could differentially tax their outputs, and use the receipts to mitigate harms they cause or facilitate.

      Democratic governments can impose differential taxes on tech companies that cause harm, impose impediments on smaller rivals, and undermine fair competition.

    2. gmascareno2028 21 May 2026
      digital status quo is allowed to continue.

      The digital status quo is characterized by tech corporations holding a substantial advantage in maintaining economic influence, continuously employing effective strategies such as upgrades, investments in new productions, and advertising to sustain their dominance.

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    academic.oup.com/book/39213/chapter/338716112
  37. ikmz-statistik.pages.uzh.ch ikmz-statistik.pages.uzh.ch
    8  Hypothesentesten – Statistik-Einführung
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    ikmz-statistik.pages.uzh.ch/begleiterin-26/08-Hypothesentesten-Grundlagen.html
  38. www.reddit.com www.reddit.com
    Had a typewriter for awhile, could use help identifying it!
    1
    1. chrisaldrich 21 May 2026

      reply to u/Novembree at https://old.reddit.com/r/typewriters/comments/1hfncyz/had_a_typewriter_for_awhile_could_use_help/

      Welcome to the Royal KMM club! Seems like lots of these have been posted in the last day including one by u/betternatured and another by u/the-other-gusta along with a very similar Royal KMG by u/Jacki-san.

      The serial number puts yours down as a KMM with an 11 inch platen manufactured in 1945. Cross reference: https://typewriterdatabase.com/royal.72.typewriter-serial-number-database

      Manual: https://site.xavier.edu/polt/typewriters/RoyalKMM.pdf

      These were really popular and ubiquitous, standard (large desktop) typewriters in the mid-century that were the workhorse of many offices. Because they were so common and so heavy, they only go for $5-25 in the used market in either unknown or marginal condition. If they're cleaned up and well-serviced they can go for more with a cap of around $300-400 depending on the level of restoration. Some with special features (like special typefaces) or provenance may go for more.

      The Royal KMM was known to have been used by writers including: John Ashberry, Harry Ashmore, Russell Baker, Ray Bradbury, Richard Bratigan, Richard Brooks, Pearl S. Buck, Johnny Carson, Norman Corwin, Frank Herbert, Ken Kesey, G.W. Lee, Harper Lee, Ursula K. LeGuin, David McCullough, Margaret Mead, Grangland Rice, and Dorothy Parker. This was also the model famously used by Angela Landsbury's character on the TV show Murder, She Wrote.

      Depending on your level of typewriter knowledge try out some of the following short films which will also provide some tips, tricks, and maintenance advice common in the era of your machine:

      Happy Typing!

      Royal KMM typewriters Royal KMM welcome
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  39. Local file Local file
    SI-B2C Contract State Transition Matrix (Interactive)
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  40. michaelrotenbergschwartz.net michaelrotenbergschwartz.net
    Love elegies. Written in the year 1732.
    3
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    michaelrotenbergschwartz.net/HammondLoveElegies.html
  41. xupsh.gitbook.io xupsh.gitbook.io
    第三章 CORDIC | FPGA并行编程
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    xupsh.gitbook.io/pp4fpgas-cn/zheng-wen/03-cordic
  42. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 19
    1
    1. sabrinaavalos2 21 May 2026

      After the hurricane, Tea Cake slowly becomes sick with rabies from the dog bite. His behavior becomes violent and paranoid. When he threatens Janie with a gun, Janie is forced to shoot him in self-defense. Tea Cake dies, leaving Janie heartbroken but forever changed by their love

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/19/
  43. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 18
    1
    1. sabrinaavalos2 21 May 2026

      A hurricane approaches the Everglades. Many people ignore the warnings, but the storm becomes deadly. Janie and Tea Cake try to escape through dangerous floodwaters. During the escape, Tea Cake saves Janie from a rabid dog but gets bitten in the process.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/18/
  44. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 17
    1
    1. sabrinaavalos2 21 May 2026

      Mrs. Turner, a mixed-race woman living in the Everglades, admires Janie because of her lighter skin and dislikes darker Black people, including Tea Cake. She tries to convince Janie to leave Tea Cake for her lighter-skinned brother. Tea Cake strongly dislikes Mrs. Turner’s prejudice.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/the-distributed-proofreaders-canada-ebook-of-their-eyes-were-watching-god-by-zora-neale-hurston-16/
  45. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 16
    1
    1. sabrinaavalos2 21 May 2026

      woman named Nunkie flirts with Tea Cake, making Janie jealous. Janie confronts Tea Cake, and the two argue but later make up. Their relationship remains strong despite moments of jealousy and insecurity

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/16/
  46. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 15
    1
    1. sabrinaavalos2 21 May 2026

      Tea Cake and Janie move to the Everglades (“the muck”), where Tea Cake works in the fields. Janie enjoys the freedom and lively atmosphere there. She becomes part of the community and experiences a simpler but happier

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/15/
  47. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 14
    1
    1. sabrinaavalos2 21 May 2026

      Janie leaves Eatonville with Tea Cake and moves to Jacksonville. People criticize her for leaving her wealthy life behind for a younger man, but Janie does not care. She chooses love and happiness over status

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/14/
  48. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 13
    1
    1. sabrinaavalos2 21 May 2026

      Tea Cake suddenly disappears for a short time, making Janie anxious. When he returns, he admits he went gambling and had a good time. Janie is upset at first, but she forgives him. Eventually, Tea Cake asks Janie to marry him, and she agrees.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/13/
  49. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 12
    1
    1. sabrinaavalos2 21 May 2026

      Tea Cake and Janie spend more time together, but Janie worries he may only want her money. Tea Cake reassures her through his actions and affection. Their bond deepens as they continue getting closer

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/12/
  50. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 11
    1
    1. sabrinaavalos2 21 May 2026

      Tea Cake continues spending time with Janie, and their relationship grows stronger. Although the townspeople gossip about him because he is younger and poorer, Janie enjoys being with him. Tea Cake makes her feel valued and happy.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/11/
  51. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 10
    1
    1. sabrinaavalos2 21 May 2026

      Tea Cake’s easygoing personality is very different from Joe Starks’ controlling behavior. He listens to Janie, makes her laugh, and includes her in activities that Joe once thought were inappropriate for women. Janie begins developing feelings for him, even though she worries about what other people might think because Tea Cake is younger and less wealthy than she is.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/10/
  52. pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
    Chapter 8
    1
    1. sabrinaavalos2 21 May 2026

      Starks becomes very sick and weak. As his health gets worse, Janie begins thinking about how unhappy she has been during their marriage. For years, Joe controlled her, silenced her, and cared more about power and appearance than her feelings.

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    pressbooks.library.torontomu.ca/theireyeswerewatchinggod/chapter/8/
  53. www.biorxiv.org www.biorxiv.org
    Zero-shot design of a de novo metalloenzyme
    1
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    biorxiv.org/content/10.64898/2026.04.23.720277v1
  54. socialsci.libretexts.org socialsci.libretexts.org
    1.1: Introduction to Positive Psychology
    1
    1. jlweber4 21 May 2026
      more!

      Flow: being really engaged and perfect level of hardness, perfect challenge, rewarding Mindfulness: focused awareness on what is going on right then - being in place, grounded in present. Learned optimism - can create joy, can control the future and life - what students need. Well being isn't just fortune, but you can do things to improve it.

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    socialsci.libretexts.org/Courses/University_of_California_Irvine/Positive_Psychology/01:_Introduction_to_Positive_Psychology/1.01:_1.1_Introduction_to_Positive_Psychology
  55. socialsci.libretexts.org socialsci.libretexts.org
    1.2: Affective Forcasting
    1
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    socialsci.libretexts.org/Courses/University_of_California_Irvine/Positive_Psychology/01:_Introduction_to_Positive_Psychology/1.02:_Affective_Forcasting
  56. aldani.quarto.pub aldani.quarto.pub
    Estrutura do ggplot2 – R4You
    1
    1. Aldani_Carvalho 21 May 2026
      expand

      Isto garante que o eixo fique encaixado na base sem espaço: scale_y_continuous( expand = c(0, 0) ) .👇 Melhor ainda é assim porque ele respeita as dismensões definidas: scale_y_continuous( limits = c(20, 40), breaks = seq(20, 40, by = 2), expand = expansion(mult = 0) )

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    aldani.quarto.pub/r4you/posts/ggplot/ggplot.html
  57. news.smol.ai news.smol.ai
    Untitled document
    7
    1. fxp007 21 May 2026
      Another secondary summary gives Humanity’s Last Exam: 64.7% vs 53.1%, possibly under different setup/effort/tool conditions.

      This is a classic example of cherry-picking data to create a narrative of superiority. By presenting a potentially non-comparable benchmark result right after a definitive one, the author casts doubt on the entire benchmarking exercise, allowing them to pick and choose the numbers that best support the 'Mythos is vastly superior' story while ignoring context.

      Data Cherry-Picking Benchmarking
    2. fxp007 21 May 2026
      Anthropic explicitly says Mythos Preview is available to launch partners in Project Glasswing, not general users... This triggered discussion of “API hoarding” and a new closed-access elite tier.

      The author frames the closed access as a reaction to a 'discussion,' but it's a deliberate corporate strategy. The term 'hoarding' is loaded and negative, whereas the article's own analysis presents it as a rational business decision. This contradiction highlights the author's attempt to have it both ways: criticizing the practice while subtly justifying it.

      Loaded Language Strategic Contradiction
    3. fxp007 21 May 2026
      The interpretation that Anthropic has “the mandate” or is undervalued at $380B is an investor thesis, not a confirmed market fact.

      This line is a critical piece of self-awareness that contradicts the article's own tone. The author, while acknowledging this is just 'investor thesis,' has spent the preceding paragraphs building the case for it, creating a hypocritical tension between the article's speculative claims and its own caveat.

      Hypocrisy Market Narrative
    4. fxp007 21 May 2026
      A key subtext in the tweets is that high-margin enterprise/coding/cyber workloads may now be sufficient to support frontier labs without broad public access to their best models. This becomes more plausible if Anthropic’s revenue is indeed compounding as fast as posters claim.

      The author presents this as a 'subtext,' but it's actually a central thesis being pushed. It reframes the 'hoarding' of powerful models not as a potential negative, but as a new, economically rational business model—a highly counterintuitive position that challenges the traditional 'open access' ethos of AI development.

      Business Model Counterintuitive Thesis
    5. fxp007 21 May 2026
      We’ve done a focused news summary run below, for those who desire more detail.

      This is a classic rhetorical device that signals the author is about to pivot away from objective reporting and into curated interpretation. The preceding text is not a 'summary' but a highly selective presentation of data points designed to support a specific thesis, making this line a disingenuous signpost.

      Rhetorical Framing Omission
    6. fxp007 21 May 2026
      If a master tactician wanted to further competitive narratives vs a potential IPO, you would be hard pressed to find a better idea than Claude Mythos... and now formally confirmed to be too dangerous to release GA, instead only restricted to 40 partners under an urgent new “Project GlassWing”

      This is a masterclass in narrative engineering. The 'too dangerous to release' claim serves a dual purpose: it creates a powerful safety narrative for Anthropic while simultaneously manufacturing scarcity and an exclusive 'private frontier' dynamic, which is a brilliant non-obvious strategic move to justify closed access and high valuation.

      Narrative Engineering Strategic Misdirection
    7. fxp007 21 May 2026
      Against the backdrop of OpenAI announcing $24B ARR, stalled ChatGPT growth and coincidental personnel moves in CEO, COO, and CMO and sensationalist rumors with CFO, this week’s events in Anthropic announcing a massive jump from $19B ARR in March to $30B ARR in April seems like a VERY strategic jab, especially considering known differences in revenue recognition, but the differential rate of growth and higher cost efficiency is undeniable… only for today to step it up a notch.

      This framing is intentionally misleading. The $30B ARR figure is not a confirmed disclosure but a market interpretation. The article's author is constructing a narrative of a 'jab' using speculative, third-party claims to build a competitive story that isn't directly supported by primary-source data from Anthropic.

      Framing Speculation
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    news.smol.ai/issues/26-04-06-anthropic-mythos
  58. Local file Local file
    Walking the talk for dementia: A unique immersive, embodied, and multi‐experiential initiative
    1

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  59. www.sec.gov www.sec.gov
    Space Exploration Technologies - S-1
    1
    1. bazalan 21 May 2026
      FORM S-1REGISTRATION STATEMENTUNDER THE SECURITIES ACT OF 1933Space Exploration Technologies Corp

      This is the day S-1 junkies have been looking forward to - everyone's favorite It Does Everything company: SpaceX! Snark is 100% intentional, mistakes or misrepresentations are not. Please don't sue.

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    sec.gov/Archives/edgar/data/1181412/000162828026036936/spaceexplorationtechnologi.htm
  60. Local file Local file
    HRL Interactive Documentation — Demo
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  61. docs.framos.com docs.framos.com
    Annotating the law | Hypothes.is
    1
    1. fjunior 20 May 2026
      Rather than ban AI or ignore it, faculty at these institutions turned to Hypothesis as a way not only to discourage over-reliance on AI tools but to challenge students to engage with deep, critical reflection on what AI produces or generates. By combining social annotation with structured assignments, they helped students become more critical readers, more thoughtful users of technology, and more confident contributors to academic conversations.

      What do you think about this?

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    docs.framos.com/en/latest/index.html
  62. max.gp max.gp
    Max Heyer - founder of enum, a European sovereign cloud
    1
    1. pyxelr 20 May 2026
      going full ai engineer, not touching code anymore
      • Shift in Role and Passion: The author has stopped writing manual code entirely after nearly two decades as a developer. They realized the actual enjoyment came from software design, architecture, and problem-solving, rather than the mechanical overhead of typing out code.
      • The "Toll" of Typing: Writing boilerplate code, null checks, imports, and repetitive logic is characterized as a "toll" paid to bring systemic ideas into reality. AI agents now handle this translation layer entirely.
      • New Core Responsibilities: The job has evolved into writing clear specifications, designing robust architectures, orchestrating multiple AI agents, and aggressively reviewing diffs to reject bad implementations.
      • The Importance of "Taste": Utilizing AI agents successfully requires profound technical taste. An engineer must understand what to insist on, detect fake test coverage, and identify load-bearing assumptions that are likely to fail.
      • Vibe-Coding Warning: Blindly relying on AI to write unread code into unverified systems results in fragile production software. Evaluating code is harder than producing it, meaning AI tools will make bad engineers worse and efficient engineers better.
      • Identity and Future Uncertainty: The author admits they would likely quit engineering altogether if forced to return to manual coding. However, they acknowledge unresolved questions regarding how this shift affects the training and hiring of junior engineers who won't build foundational muscle memory.

      Hacker News Discussion

      • The Skill Disconnect for Juniors: A dominant theme is how junior developers will gain the necessary "taste" and evaluation skills if they completely skip the grueling phase of writing and debugging code manually.
      • The Cognitive Load of Code Review: Many commenters argue that reading, auditing, and maintaining AI-generated code is mentally exhausting. They note that debugging subtle, hallucinated logic errors written by an agent is often more difficult than writing the logic from scratch.
      • Loss of Mastery and Dependency: Users express concern over the degradation of raw coding skills. Becoming entirely reliant on a fluctuating AI tool stack risks leaving engineers stranded if the quality of the models regresses or changes.
      • Analogy to Higher-Level Languages: Several participants view this evolution as a natural continuation of computer science history, comparing the shift to moving from Assembly to C, or from C to Python, where engineers routinely surrendered low-level control for higher abstraction.
      AI programming VibeOps
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    max.gp/writing/going-full-ai-engineer-not-touching-code-anymore/
  63. www.wheresyoured.at www.wheresyoured.at
    AI Is Too Expensive
    1
    1. pyxelr 20 May 2026
      AI Is Too Expensive
      • Fundamental Economic Unviability: AI is currently financially unsustainable for everyone except hardware manufacturers (like NVIDIA) and construction firms benefiting from data center buildouts.
      • Astronomical Capex Sunk Cost: Hyperscalers (Microsoft, Google, Meta, Amazon) have spent over $800 billion in the last three years, with trillions more planned through 2027. To break even or justify this, they would need unprecedented, multi-hundred-billion-dollar surges in AI-specific revenue that are nowhere in sight.
      • Obscured AI Revenue: Tech giants consistently hide actual AI revenues within broader categories. Traded companies rely on "revenue run rates" (which are monthly snapshots, not true annual revenues) to project false stability.
      • Heavy Dependency on OpenAI and Anthropic: Over 50% of hyperscalers' revenue backlogs (Remaining Performance Obligations) are driven directly by OpenAI and Anthropic—unprofitable entities that burn billions in compute and require massive cash injections just to survive.
      • Exploding, Unpredictable Customer Costs: Enterprise clients (such as Zillow and Stripe) are burning through annual token budgets in mere months due to executive mandates to "use AI for everything."
      • Lack of Transparency and Accountability: AI labs like Anthropic do not provide standard corporate service-level agreements (SLAs) or granular usage telemetry. This makes it virtually impossible for enterprise customers to predict or manage token expenditures.
      • Zero Measurable ROI: The heavy adoption of AI inside companies is creating structural chaos and technical debt. It relies entirely on experimental token spending driven by corporate fear of missing out (FOMO) rather than actual productivity gains.

      Hacker News Discussion

      • Audience Capture vs. Solid Reporting: Some commenters argue that the author has fallen into "audience capture," catering heavily to a crowd that wants to see AI fail. Conversely, defenders point out that he uncovers crucial insider metrics and that tech companies have historically hidden weak business margins behind hype.
      • The Reality of Compute Constraints: Users debate whether the market is truly saturated or experiencing a massive supply crunch. Providers are routinely hitting capacity limits, with backlogs growing into the hundreds of billions of dollars.
      • Unsustainable Investment vs. Technology Value: Multiple comments draw a distinct line between AI being a valuable tool and the current investment levels being a bubble. Many believe AI will face a "race to the bottom" where providers operate at a loss until prices drop significantly.
      • Local and Open Source Alternatives: Some argue that because strong models can now be run locally for free, or trained cheaply by international competitors, the expensive hosting models of major AI labs face an uphill battle to ever turn a profit.
      AI FinOps
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  64. thetorontobeachclub.com thetorontobeachclub.com
    Toronto Beach Club
    5
    1. vamanda 20 May 2026
      Indulge in seasonal buffet brunch.

      Good Practice (Understandable) Simple navigation/menu structure

      The website has a straightforward navigation menu with predictable categories such as dining, reservations, and events. This supports the “Understandable” principle because users can easily predict where information is located without confusion or cognitive overload.

    2. vamanda 20 May 2026
      ntertainment is built into the experience, not an afterthought

      Bad Practice (Robust / Screen Readers Decorative images and multimedia sections The homepage relies heavily on visual imagery and multimedia content. If these images or interactive elements are not properly labeled for assistive technologies, screen readers may not communicate the content effectively to visually impaired users.

    3. vamanda 20 May 2026
      SEE YOU AT THE BEACH

      The website supports keyboard navigation using the Tab key, which improves accessibility for users who cannot use a mouse due to motor impairments. This follows the WCAG “Operable” principle because users can move through menus and interactive elements without relying on mouse hovering alone.

    4. vamanda 20 May 2026
      Our dynamic beach club experience is founded on the art of entertaining. From weddings and milestone celebrations to corporate gatherings and brand events, every detail is designed to transport your guests beyond the ordinary. With versatile spaces overlooking the shoreline and a setting that shifts seamlessly from day to night, Toronto Beach Club becomes more than a venue. It’s a destination where moments are created, memories are shared, and every occasion feels like an escape.

      Bad Practice (Colour Contrast)Light text over image backgrounds. Some text appears over bright photographic backgrounds, which may create low colour contrast. This can make reading difficult for users with low vision or colour blindness.

    5. vamanda 20 May 2026
      Where the beach is yours, no membership required

      This is good practice. The homepage uses large headings and visually clear sections, which helps users perceive information more easily. This follows the perceivable principle because content is easier to distinguish and read for users with visual impairments.

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    thetorontobeachclub.com/
  65. Local file Local file
    Untitled document
    1
    1. RealDidacticus 20 May 2026
      I started undoing the 'participatory' design plans I unilaterally made to reconceive acollective methodology with more uncertain, voluntary, and relational dynamics. Surprisingly, this'ineffective' ongoing turn became a strength rather than a limitation—

      No plan is unilateral, we are a conduit of past relationships, of people who have influenced us, and we acknowledge this so much that we allow the transference of autonomy through "differently abled" people guardians, stewards of nature, animal caretakers, and political representatives.

      What Volpi is looking for without stating is a sustainable equalised economy where there are no power monopolies and notable hierarchies that may lead to oppression. But to say that "inefficiency" is a "strength" unkowingly perpetuates those oppressive structures, because this "inefficiency" is almost exclusive of wealthy people. Volpi's language is colonised, as they probably don't realise this.

      Say they get involved in an actually slow process, one where they don't propose, but wait for others to ask, one, like an ethnography, where they learn and listen and don't try to impose themselves and their ideas because that is the productivist system that academia perpetuates... then the group they end up in will either be a marginally small group of outcast people, or privileged (or both), with minimal potential impact for change; or they will end up in a bigger already existing association where they in a way "inflitrate" and only over multiple years start to achieve trust capital to push their ideas (having also taken some others' in order to claim epistemic humility and a certain representativeness).

      Let's see... how do I spell this? We must not condone infinite growth, but when it comes to things like ending poverty, I think our stance should be unambiguously clear that this is progress, that this is positive.

    Annotators

  66. math.libretexts.org math.libretexts.org
    15.1: Inflation
    3
    1. ravsar 20 May 2026
      when the prices of produce rise in the winter, we don’t call this inflation because prices typically decrease again in the spring

      Price of a single product is irrelevant. You mean seasonal influences?

    2. ravsar 20 May 2026
      The value of your bank balance also decreases because, with higher prices, it takes more money to purchase the same quantity of goods and services.

      Why is this sentence needed? Deposits are money.

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    math.libretexts.org/Workbench/Columbia_College_Chicago_Math_Texts_and_Ancillaries/15:_Finance/15.01:_Inflation
  67. www.ox.ac.uk www.ox.ac.uk
    Why is almost everyone right-handed? The answer may lie in how we learned to walk
    1
    1. pyxelr 20 May 2026
      Why is almost everyone right-handed? The answer may lie in how we learned to walk
      • Human handedness has long been an evolutionary enigma, with roughly 90% of people across all cultures preferring their right hand—a population-level bias not found on this scale in any other primate species.
      • A new study led by the University of Oxford and published in PLOS Biology suggests that human right-handedness is tied to two defining evolutionary traits: bipedalism (walking on two legs) and brain expansion.
      • Researchers analyzed data from 2,025 individuals across 41 primate species. When factoring in brain size and the relative length of arms to legs (an anatomical marker of bipedalism), humans no longer appeared as an evolutionary anomaly in the models.
      • The findings support a two-stage evolutionary process:
        • First, walking upright freed the hands from locomotion, creating selective pressure for specialized, lateralized manual behaviors.
        • Second, as the brain dramatically expanded and reorganized, this rightward bias solidified into the near-universal pattern seen today.
      • Evolutionary projections of extinct human ancestors suggest a gradient: early hominins (Ardipithecus and Australopithecus) likely had only a mild rightward preference, which strengthened with the genus Homo (Homo ergaster, Homo erectus, and Neanderthals) before reaching the modern extreme in Homo sapiens.
      • Homo floresiensis (the small-brained "hobbit" species) is a striking exception with a much weaker predicted preference, aligning with its smaller brain and body adapted to a mix of climbing and walking.

      Hacker News Discussion

      • Cooperation vs. Competition Dynamics: Commenters noted that population-level handedness may stem from the collaborative nature of humans, where learning tasks is easier when using the same hand. Conversely, in purely competitive environments like ping-pong or fencing, a 50-50 split or a higher prevalence of left-handedness emerges because lefties enjoy the evolutionary "frequency-dependent" advantage of being rare and unpredictable to opponents.
      • Linguistic and Cultural Asymmetry: A discussion arose regarding how the word "right" historically equates to correctness, law, or justice in various Indo-European languages, while "left" often holds negative connotations (e.g., originating from words meaning "weak" or Latin roots like sinister). Users debated whether this linguistic baggage is uniquely Western or reflects an inherent human bias toward pairing up/light/right with positivity.
      • Innate vs. Learned Handedness: Users shared personal anecdotes about learning to use their non-dominant hand for complex tasks, such as switching from right-handed arrow keys to left-handed WASD controls in PC gaming, or playing musical instruments.
      • Adaptability of Left-Handed Individuals: Left-handed users emphasized that living in a predominantly right-handed world forces them to be functionally ambidextrous to navigate daily tools, trackpads, and vehicle stick shifts, whereas right-handed individuals rarely have to adapt their non-dominant hand unless prompted by injury.
      evolution anthropology bipedalism handedness
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    ox.ac.uk/news/2026-05-15-why-is-almost-everyone-right-handed-the-answer-may-lie-in-how-we-learned-to-walk
  68. wkontenerach.pl wkontenerach.pl
    Współdzielenie Skills i Agents między Codex i Claude Code
    1
    1. pyxelr 20 May 2026
      Współdzielenie Skills i Agents między Codex i Claude Code
      • The Problem: Developers using multiple local AI terminal agents (such as Codex, Claude Code, or OpenCode) quickly face fragmentation when trying to manage custom skills, agent roles, and project-specific instructions. Files end up being scattered across varying default directories or duplicated manually across the user's home folders.
      • The Solution: A centralized directory architecture within the project repository that acts as a single source of truth (ai/), sharing identical configurations across different AI tools through local symbolic links (symlinks).
      • Directory Layout & "Source of Truth":
        • All active configuration files reside inside a single /ai folder, split into /ai/agents (who the model should be—e.g., Architect, Reviewer, Incident Commander) and /ai/skills (how the model performs tasks—e.g., API Review, Security Check, Frontend QA).
      • The Symlink Mechanism:
        • Instead of configuring generic home directories (~/.claude or ~/.codex), local tool-specific directories are generated inside the project (.agents/ for Codex and .claude/ for Claude Code).
        • Using terminal commands (like ln -sfn on macOS/Linux or New-Item -ItemType SymbolicLink on Windows PowerShell), symlinks are established to point both .agents/ and .claude/ folders to the exact same /ai sub-directories.
      • Key Advantages:
        • Centralization: Establishes a single, distinct source of truth for all AI interactions within the workspace.
        • Tool Compatibility: Seamlessly supplies the exact same data to different AI agents without manual file copying.
        • Team Portability & Version Control: Because Git natively tracks symbolic links, the entire team receives the exact same AI tooling, workflows, and prompts directly upon cloning the repository.
      AI Claude Copilot Codex programming polish
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    wkontenerach.pl/wspoldzielenie-skills-miedzy-codex-i-claude-code/
  69. www.biorxiv.org www.biorxiv.org
    The functional maturation and capacitation of mouse spermatozoa is underpinned by global remodeling of the cellular phosphoproteome
    4
    1. EMBOpress 20 May 2026

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

      Learn more at Review Commons

      Reply to the reviewers

      Manuscript number: RC-2025-03227R

      Corresponding author(s): Dr. David Skerrett-Byrne & Prof. Brett Nixon

      1. General Statements

      We are grateful to the reviewers and editorial team for their thoughtful and constructive evaluation of our manuscript. The comments provided were insightful and have substantially strengthened the rigor, clarity, and presentation of the study. In response, we have carefully revised the manuscript throughout, including clarification of conceptual interpretations, expansion of methodological detail, refinement and condensation of the Discussion, as well as addition of new supplementary analyses and figures. Collectively, we believe these revisions have improved both the transparency and accessibility of the work while reinforcing the central conclusions of the study.

      At its core, this study sought to address a major unresolved question in reproductive biology: how spermatozoa, which are transcriptionally and translationally inert, achieve functional competence during post-testicular maturation. Using deep, stage-resolved phosphoproteomics integrated with functional validation approaches, we demonstrate that the majority of sperm phosphoproteomic remodelling occurs during epididymal maturation rather than during capacitation, challenging long-standing paradigms in the field. Beyond generating one of the deepest sperm phosphoproteomic resources currently available (>14,000 phosphosites), the study also provides functional and physiological context through kinase inhibition studies, in vivo knockout phenotypes, and the development of the ShinySpermPhospho online resource to facilitate community access and future discovery.

      Importantly, through the review process we have worked carefully to ensure that the manuscript more clearly distinguishes data-driven conclusions from hypothesis-generating interpretations, particularly in areas relating to kinase prediction, metabolic regulation, and phosphoproteomic remodelling. We believe the revised manuscript now presents a more balanced and rigorous framework while preserving the significance of the central findings.

      Overall, we hope the revised manuscript now provides a valuable resource and conceptual advance for the reproductive biology community, with implications extending from fundamental sperm cell biology to translational opportunities in male infertility and contraceptive development.

      2. Point-by-point description of the revisions

      REVIEWER #1

      The manuscript by Skerrett-Byrne and collaborators represents a comprehensive and technically sophisticated phosphoproteomics study. Using high-resolution mass spectrometry on mouse sperm obtained from the caput and cauda regions of the epididymis, both before and after capacitation, the authors generated a more complete database of phosphorylation changes in these cells. One of the most interesting outcomes is that most of these changes occur during sperm maturation, rather than sperm capacitation. The work is important and relevant, and the information obtained could be valuable for reproductive biologists working in basic science, as well as for the identification of novel contraceptive targets.

      __Answer: __We thank the reviewer for their positive assessment of our work and for recognising the value of the datasets we have generated for supporting future innovations in both fundamental reproductive biology and the identification of novel contraceptive targets. We are also delighted that the reviewer has recognised the significance that, contrary to previously thought, the majority of the phosphorylation changes we report occur during epididymal maturation, rather than subsequently during capacitation.

      • The title should include a reference to sperm capacitation, as most of the study focuses on comparisons between epididymal maturation and capacitation, and the functional experiments are based on the latter. __Answer: __We thank the reviewer for this suggestion and have revised the title to reflect the importance of our focus on both phases of post-testicular sperm maturation, namely epididymal sperm maturation and sperm capacitation (please see line 1).

      • Considering the newly reported changes in phosphosites, it would be desirable to include validation at the individual protein level for at least a few examples, using an independent technique such as western blotting. __Answer: __We thank the reviewer for this thoughtful suggestion and fully appreciate the motivation to seek orthogonal validation of phosphoproteomic findings. However, we respectfully wish to express our reservations regarding the use of antibody-based validation of site-specific phosphorylation events, a technique that is increasingly being recognised as problematic and, in many cases, less reliable than modern MS-based approaches (Nature, PMID: 39506148). Indeed, high-resolution mass spectrometry provides direct, site-resolved identification and quantification of phosphorylation events with substantially greater specificity, accuracy, and proteoform resolution than antibody-based methods. For this reason, MS-based phosphoproteomics is now widely regarded as the gold standard for mapping phosphorylation dynamics.

      With regard to the use of antibodies, many commercially available phospho-specific antibodies lack sufficient site specificity, often have poorly defined or undocumented epitope recognition, and frequently fail to discriminate between closely related proteoforms or neighbouring phosphorylation sites. Indeed, recent large-scale evaluations have demonstrated that many widely used antibodies do not reliably bind their intended targets, raising concerns about reproducibility and interpretability across the biomedical sciences (PMID: 37995198). In one study, testing the utility of >600 antibodies, two thirds failed to work as described (PMID: 37995198), while the literature also features other studies (e.g. PMID: 31612854) reporting that certain antibodies (SC-138763) do not bind their stated target despite having been "used in 15 published manuscripts to ascribe specific properties to the protein in normal and disease states", collectively cited >3,000 times.

      Accordingly, while we recognise the importance of independent validation, we contend that antibody-based validation may not be the most appropriate strategy to improve the robustness of the conclusions in this study. It is for this reason that we elected to strengthen confidence in our findings through multiple complementary approaches, including rigorous statistical filtering, extensive in-silico pathway and kinase analyses, selective pharmacological inhibition of target proteins, and in vivo functional interrogation using knockout mouse models. Together, these orthogonal strategies provide additional biological validation linking the reported phosphorylation changes to aspects of sperm function.

      We have clarified this rationale in the revised manuscript and briefly expanded the discussion to touch on these methodological strengths and limitations (please see lines 754 - 759).

      • In the knockout models, it is not possible to distinguish between defects in spermatogenesis and those arising during maturation or capacitation. A parameter directly related to spermatogenesis should therefore be included, for example, testicular weight or histology, sperm number, and sperm morphology. __Answer: __We thank the reviewer for raising this important point. We agree that systemic knockout models do not allow definitive discrimination between defects arising during spermatogenesis versus those occurring downstream during post-testicular sperm maturation or capacitation. Unfortunately, the additional parameters suggested by the reviewer do not form part of the standardised phenotyping pipeline implemented by the International Mouse Phenotyping Consortium (IMPC) and the European Mouse Mutant Archive (EMMA). As such, these data are not available for the knockout lines examined in this study and cannot be retrospectively generated. We have therefore clarified this limitation more explicitly in the revised manuscript and have framed the knockout data as physiological validation concerning the functional relevance of the parent protein rather than as definitive evidence of stage-specific or phosphorylation-dependent mechanisms of action. Importantly, the consistency of impaired sperm motility and fertilisation outcomes across multiple independent knockout lines supports the biological importance of the parent proteins identified, while acknowledging that the precise developmental window of their action remains to be resolved. While we regrettably concede that it is beyond the scope of this study, we do acknowledge that future studies will be required to dissect these mechanisms with greater resolution, ideally using germ cell-specific or temporally controlled knockout models, or targeted manipulation of key phosphoproteins and/or their phosphorylation motifs. Such approaches will be essential if we are to be able to distinguish roles of target proteins in spermatogenesis from those that occur downstream during epididymal maturation and capacitation (please see lines 725 - 733).

      • Error values, sample size, and statistical analyses are missing from Figure 7 and should be provided for clarity. __Answer: __We apologise for this omission and have now updated Figure 7 and its legend to include sample sizes, error values, and details of the statistical analyses used, thereby improving clarity and reproducibility of these data.

      In addition, we have clarified that sperm functional data derived from EMMA knockout lines are generated from cryopreserved samples comprising pooled cauda epididymal spermatozoa collected from 10 heterozygous males per line (PMID: 17709347, 38839949). As such, each data point represents a pooled biological sample, consistent with standardised EMMA/INFRAFRONTIER protocols (PMID: 25414328, 27262858, 38839949). Where appropriate, we have also included additional reproductive metrics at the level of IVF cycle (where available) and individual litters, including average litter size and fetal sex distribution (with exceptions for specific lines where such data are not available). These details are now captured in both the Methods and relevant figure legends (please see lines 453 - 457, 1228 - 1234, 1431 - 1434, 1533 - 1536, 1572 - 1575, Figure 7 & S6).

      REVIEWER #2

      In this manuscript, the authors examine dynamic modifications of the sperm phosphoproteome during epididymal transit and capacitation. They compare three distinct populations differing in anatomical localization and activation status: caput sperm, non capacitated cauda sperm, and capacitated cauda sperm. Using high resolution tandem mass spectrometry, they reveal that phosphorylation changes during epididymal passage are far more extensive than previously appreciated. These findings are further validated in genetically modified animal models, where disruption of selected genes encoding for phosphoproteins results in marked defects in sperm motility and fertilization capacity.

      __Answer: __We thank the reviewer for their positive and thoughtful evaluation of our study and for recognising both the depth of the phosphoproteomic dataset and the importance of the functional validation experiments; sentiments that we whole heartly agree with.

      • Throughout the text, and particularly in the paragraph entitled 'Epididymal maturation accounts for the majority of maturation associated sperm cell signaling,' it seems that phosphorylation is interpreted as inherently activatory and dephosphorylation as inhibitory (lines 248-252). Since this relationship is not universally applicable, it would be valuable to address this issue at the outset of the paragraph and to discuss how phosphorylation events are context dependent in their effects on protein function. __Answer: __We thank the reviewer for highlighting this important conceptual point. We fully agree that phosphorylation is not inherently activatory, nor is dephosphorylation necessarily inhibitory, and that the functional consequences of phosphorylation are highly context dependent. We have revised the indicated paragraph to explicitly acknowledge this at the outset to ensure that phosphorylation changes are interpreted as regulatory rather than intrinsically directional (please see lines 219 - 222).

      • Lines 392-393: the claim that "the introduction of each inhibitor to populations of capacitating spermatozoa led to a significant reduction..." is not fully supported by data and should be toned down. In fact, two out of three inhibitors, do not significantly affect the acrosome reaction. __Answer: __We thank the reviewer for this careful assessment and agree that the original wording overstated the nuances of the effects of individual inhibitors. We have revised the text to explicitly report the corresponding p-values and to distinguish between statistically significant and non-significant trends. Specifically, inhibition of PAK1 produced a statistically significant reduction in the acrosome reaction, whereas inhibition of STK33 (p = 0.0574) and HIPK4 (p = 0.0911) resulted in consistent, but non-significant, reductions. Importantly, combined inhibition of all three kinases yielded a robust and statistically significant suppression of acrosomal exocytosis. The revised wording now accurately reflects the quantitative data (please see lines 419 - 424, 700 - 703).

      • The discussion section, spanning 11 pages, is overly long and contains considerable repetition. I recommend transferring the detailed description of experiments to the 'Results' section and using the discussion primarily to synthesize and highlight the novel findings while limiting speculative content. For example, the content in lines 509-530 could be condensed and relocated to the Results. Likewise, other detailed examples would be more appropriately presented within their respective result paragraphs. __Answer: __We thank the reviewer for this constructive feedback. We agree that the Discussion was overly long and on reflection does contain some unnecessary repetition. In response, we have substantially condensed the Discussion (shorten by 641 words), relocated and shorten detailed descriptions of experimental observations to the Results section where appropriate (including the suggestion made), and focused the revised Discussion on synthesis of the key findings and their broader implications. We should note, to address certain review comments, this require further additions to the discussion but we have endeavour to keep this brief (please see lines 309 - 326 and throughout the discussion).

      • Minor points:

      • To improve reproducibility, the suppliers of all reagents should be specified together with their catalogue numbers
      • Figure 7: it is unclear which data are statistically significant
      • Figure 7B: fertilization capacity should be assessed at an earlier stage, as the cleavage rate to 2-cell embryo may be affected by factors unrelated to the sperm ability to fertilize

      __Answer: __We thank the reviewer for these suggestions. We have now added supplier information and catalogue numbers for all reagents to the Methods section to improve reproducibility (please see lines 1256 - 1257, 1295, 1299, 1310, 1333 - 1334, 1343 - 1344, 1388 - 1389, 1401, 1406). We have revised Figure 7 and its legend to clearly indicate statistically significant differences, including sample sizes and statistical tests used. Lastly, we agree that assessment at earlier fertilisation stages would complement our featured assessment of sperm fertilisation competence. Regrettably, all IVF data were generated via standardised and unbiased IMPC/EMMA pipelines. As such, cleavage rate to the 2-cell stage represents the earliest uniformly available endpoint across all knockout lines. We have clarified this limitation in the revised manuscript (please see lines 723 - 724).

      REVIEWER #3

      This is technically sophisticated phosphoproteomic study of mouse sperm maturation across the epididymis and during capacitation. The dataset is deep (>14,000 phosphosites) and the analyses integrate high-resolution MS, immunofluorescence, IPA, kinase mapping, pharmacological inhibition, and knockout mouse models. The manuscript represents a nice resource for the field. However, several issues limit clarity, mechanistic interpretation, and robustness of the conclusions. In particular, the manuscript's scope is extremely large, making some conclusions insufficiently supported, and some analyses require better control, methodological transparency, or deeper mechanistic connection. It gives the impression that some mechanistic data was added to descriptive data in order to increase the manuscript's impact, although the current mechanistic data is not convincing.

      Major concerns

      1. Conceptual Overreach - "Epididymal maturation accounts for 86% of phosphorylation changes" The manuscript repeatedly emphasizes that epididymal maturation causes the majority of phosphoproteomic remodeling. While the data indeed show large quantitative differences, several conceptual issues remain:

        • The caput vs. cauda comparison includes differences in protein abundance, not only phosphorylation*
        • Many phosphosites lost in the cauda may reflect protein loss, not dephosphorylation (the authors acknowledge this, but quantitative controls are insufficient)*
        • The normalization method for phosphopeptide abundance vs total protein abundance is needed*
        • It is unclear whether the same amount of starting material and equal protein loading were used across stages I would suggest to perform (or explicitly describe) normalization using matched proteome intensities. Provide supplementary plots showing phosphosite/parent-protein normalization to avoid overinterpreting phosphosite loss as dephosphorylation*

      __Answer: __We thank the reviewer for this important and constructive critique and agree that interpretation of phosphoproteomic changes during epididymal maturation must carefully consider concurrent remodelling of the underlying sperm proteome.

      To directly address the concern that phosphosites lost in the cauda may reflect protein loss, not dephosphorylation, we have now explicitly compared these phosphoproteins lost during caput-to-cauda transit with proteins shown to be lost or reduced over the same maturation window in a previously published matched proteomic analysis of the same sperm populations. This comparison revealed that 527 phosphoproteins, out of a total of 966 phosphoproteins lost, overlapped with proteins lost during epididymal maturation, while a further 88 phosphoproteins aligning with proteins exhibiting reduced abundance during transit. While these data indicate that a subset of phosphosite loss can be attributed to complete loss of the parent protein, the remaining phosphoproteomic changes (45.4%) cannot be fully explained by protein disappearance alone and are therefore consistent with extensive phosphoproteomic remodelling. We have documented this information in a new panel of Supplementary Figure 1 (Figure S1B) and the corresponding text has been revised accordingly (please see lines 182 - 188).

      With respect to normalisation strategies, we respectfully note that normalisation of phosphopeptide intensities to total protein abundance is not universally accepted in large-scale phosphoproteomic analyses (PMID: 30190555, 34857927, 38576152), particularly in systems undergoing extensive proteome remodelling such as maturing spermatozoa. In many contexts, including our own previous work, phosphoproteomic analyses are performed on equal protein input and interpreted at the level of phosphopeptide abundance, with functional relevance established through orthogonal biological validation rather than ratio-based correction to total protein levels.

      Lastly, all samples in this study were diluted to equal total protein amounts prior to phosphopeptide enrichment, ensuring consistent input material across all sperm populations (originally noted in the manuscript, please see line 1339). We have now clarified this explicitly in the Results section to ensure this is not missed (please see lines 146 - 147). Moreover, our conclusions are supported by independent in-silico analyses, pharmacological inhibition studies, and in vivo knockout models, collectively providing functional validation that extends beyond phosphosite quantification alone.

      Finally, to address concerns regarding potential conceptual overreach, we have revised the language surrounding the statement that epididymal maturation accounts for ~86% of phosphorylation changes to ensure precise interpretation. Specifically, we have clarified that this value refers to the proportion of statistically significant differences in phosphopeptide abundance detected across maturation stages, to avoid implying direct measurement of net enzymatic dephosphorylation (please see lines 519 - 520).

      Importantly, having addressed the reviewer's concerns detailed above, we believe the data do support the conclusion that the majority of sperm phosphoproteomic remodelling occurs during epididymal maturation rather than during capacitation. While we have tempered our language to improve clarity, the central quantitative observation that epididymal transit represents the dominant phase of phosphoproteomic remodelling remains supported by the revised analyses.

      • Capacitation analysis is underpowered and oversimplified*

      The authors state that capacitation leads to "modest" changes. However:

        • The capacitation protocol uses dibutyryl-cAMP + pentoxifylline, which may bypass early physiological signaling. This is a important red flag __Answer: __We thank the reviewer for this important point and agree that the choice of capacitation conditions influences the nature and magnitude of signalling events detected. The use of dibutyryl cAMP and pentoxifylline represents a well-established and widely adopted experimental model to induce robust and synchronised capacitation-associated signalling in mouse spermatozoa, acting specifically via the activation of the canonical cAMP/PKA signalling axis (PMID: 36384108, 22458710, 16221991). While we acknowledge that this approach bypasses some upstream physiological signalling events that initiate capacitation during sperm transit of the female reproductive tract, it is intentionally employed to provide a reproducible capacitation stimulus, specifically enabling us to discriminate phosphorylation changes associated with the attainment of sperm fertilization competence. This strategy also directly addresses a limitation of working with mouse spermatozoa in which these cells rapidly succumb to cell senescence/death within a matter of ~1-3 hours in an in vitro* setting. In our previous studies, we have noted that this time period is insufficient to achieve high levels of capacitation among populations of mouse spermatozoa, unless pharmacological agents (i.e. dibutyryl-cAMP + pentoxifylline) are supplemented to accelerate capacitation (PMID: 15252132). This is now a widely accepted paradigm in the field and one that enables us to deliver on our stated objective of assessing the phosphorylation status of fertilization competent spermatozoa, as opposed to those that are captured during early phases of the capacitation cascade.

      Importantly, our conclusion that capacitation is associated with comparatively fewer phosphoproteomic changes is based on direct quantitative comparison with epididymal maturation under identical analytical conditions, and is not intended to downplay the biological importance of this critical maturation event. Even under the capacitation-inducing conditions employed herein, the scale of phosphoproteomic remodelling observed was substantially smaller than that occurring during epididymal transit, underscoring the influence of epididymal maturation over the status of the sperm phosphoproteome.

      To address this concern, we have revised the manuscript to clarify that the capacitation-associated phosphoproteomic changes reported here are specific to the experimental model used and likely represent a conservative estimate of signalling complexity under physiological conditions. We have also tempered language implying generalisation beyond this context (please see lines 329 - 332, 488 - 491, 673 - 677).

      • *

      • Kinase prediction and functional validation require more rigor*

      The identification of 343 kinases that may regulate phospho-changes is extremely broad. Issues:

        • The kinase-substrate assignments rely heavily on in silico predictions (IPA, PhosphoSitePlus), which often contain non-sperm data. __Answer: __We thank the reviewer for this important observation and fully agree that kinase-substrate assignments inferred from in-silico* resources such as IPA and PhosphoSitePlus are largely derived from non-sperm systems and therefore must be interpreted cautiously.

      Importantly, this limitation reflects a broader and well-recognised gap in the field; regrettably comprehensive, experimentally validated kinase-substrate networks do not currently exist for mammalian spermatozoa on this scale, particularly in the context of epididymal maturation and capacitation. The primary objective of the present study was therefore not to define definitive kinase-substrate relationships, but to generate a high-depth, sperm-specific phosphoproteomic resource that can serve as a foundation for hypothesis generation and future mechanistic interrogation.

      Accordingly, in-silico kinase prediction tools were employed to contextualise the phosphoproteomic data and to prioritise candidate kinases for functional testing, rather than to assert sperm-specific kinase-substrate specificity. We have revised the manuscript to clarify that these predictions represent informed starting points in a system where such information is currently lacking, and that functional relevance was subsequently assessed using complementary pharmacological and genetic approaches (please see lines 383 - 388, 682 - 686).

      By providing a deep, stage-resolved phosphoproteomic dataset encompassing more than 14,000 phosphosites, this study establishes a much-needed reference framework for the reproductive biology community, enabling future targeted validation of kinase-substrate relationships in sperm. We believe this resource-based contribution represents a major strength of the work and addresses a critical knowledge gap in the field.

      • *

        • Please explain the rationale by which, from 343 candidate kinases, 3 (STK33, HIPK4, PAK1) are selected.*
        • The pharmacological inhibitors used have off-target effects (ML281 inhibits multiple CMGC kinases; Foretinib inhibits MET/VEGFR; NVS-PAK1-1 inhibits PAK1/2/3).*
        • No control experiments are included to confirm kinase inhibition in sperm (e.g., phosphosite-specific Western blots)* __Answer: __We split this comment from the above, to best address this important critique. We agree that kinase-substrate relationships inferred from phosphoproteomic data must be interpreted with caution. The identification of 343 kinases in this study was not intended to represent a definitive catalogue of all sperm-specific kinase-substrate interactions, but rather to provide insights into kinases that potentially contribute to phosphoproteomic remodelling of mouse spermatozoa during the different phases of their post-testicular maturation. These kinases were identified through integration of multiple complementary approaches, including direct detection within the phosphoproteome, upstream regulator prediction using IPA, curated kinase-substrate databases, and comparison with previously published epididymal sperm proteomes.

      From this broader resource, we deliberately restricted functional interrogation to a small subset of kinases putatively associated with capacitation-induced phosphoproteomic changes. STK33, HIPK4, and PAK1 were selected based on their predicted association with capacitation-specific phosphorylation events, representation across distinct kinase families, lack of prior functional characterisation in terms of either sperm maturation or function, and availability of well-characterised pharmacological inhibitors suitable for functional perturbation. We fully acknowledge that the inhibitors employed are not absolutely kinase-specific and may exhibit off-target effects. Accordingly, we have revised the manuscript to clarify that these experiments are intended to test functional dependence on kinase activity rather than to establish direct kinase-substrate relationships. The observation that combined inhibition of three mechanistically distinct kinases produced a robust and additive suppression of the acrosome reaction supports the conclusion that kinase activity is required for this process, while avoiding overinterpretation of individual kinase specificity.

      We have revised the language throughout the manuscript to more clearly reflect these limitations and to frame the kinase inhibition experiments as functional validation of phosphoproteomic predictions rather than definitive mechanistic proof (please see lines 383 - 388, 405 - 406, 682 - 686, 705 - 710).

      • *

      • *

      • The knockout-mouse validation section is underdeveloped*

      The linkage of KO phenotypes to phosphorylation changes is potentially powerful but currently weak.

      Issues:

        • Most KOs are systemic deletions, not sperm-specific; phenotypes could stem from developmental defects.*
        • Some proteins validated (e.g., ACO2, CMPK1) regulate core metabolism; their phenotypes may not reflect phosphoregulation but loss of essential protein function.*
        • No evidence is provided that the KO affects the specific phosphosites detected in the MS dataset.* __Answer: __We thank the reviewer for this important clarification. We agree that since the knockout models employed represent systemic deletions, they cannot directly resolve sperm-specific or phosphosite-specific mechanisms, and it was not our intention to suggest otherwise. We have revised the manuscript to explicitly frame the knockout phenotypes as evidence of physiological relevance of the identified phosphoproteins, rather than as direct validation of individual phosphorylation events (please see lines 723 - 734).

      We further clarify that for proteins with central metabolic roles, the observed phenotypes likely reflect loss of essential protein function rather than isolated disruption of phosphoregulation. Accordingly, we have tempered our language and emphasise that these data support functional importance while highlighting the need for future studies employing germ cell-specific or phosphosite-targeted models (please see discussion).

      • *

      • Immunofluorescence and Western blots need improved quantification*

      Figures showing PKA substrate, pY, pT, pS changes are visually compelling but lack:

        • quantification across biological replicates*
        • explanation of antibody specificity (e.g., pan-PKA sites include RRXS/T motifs; cross-reactivity possible). __Answer: __We thank the reviewer for this comment and appreciate the emphasis on rigor in antibody-based analyses. We would like to clarify that the immunofluorescence and immunoblot data presented in this study do include densitometric based quantification taking into account data generated from three independent biological replicates. This is indicated by the inclusion of error bars and as stated in the relevant figure captions. (please see lines 1164 - 1168 "All immunoblotting experiments were repeated with at least three biological replicates. Densitometric data normalization was performed against the loading control protein GAPDH, and each value subsequently expressed as a fold change relative to the caput sperm. Data were analyzed by one way ANOVA with GraphPad Prism.).*

      With respect to antibody specificity, we fully agree that phospho-specific and motif-based antibodies have inherent limitations, including epitope ambiguity and the inability to resolve site-specific phosphorylation with amino acid precision (please see our answer to Comment #2 from Reviewer #1 above). For this reason, the antibodies employed here represent well-established, widely used markers in sperm biology and were included to illustrate global phosphorylation trends rather than to validate individual phosphosites. Importantly, quantitative and site-resolved interpretation of phosphorylation dynamics throughout the manuscript is derived from mass spectrometry based phosphoproteomics, which provides substantially greater specificity and resolution than antibody-based approaches.

      • *

      • Many interpretations about metabolism, storage, oxidative stress, and quiescence are speculative*

      The discussion provides attractive models linking phosphorylation to:

        • suppression of glycolysis*
        • quiescent metabolic state in cauda epididymis*
        • activation of antioxidant pathways*
        • UPR and proteostasis modifications However, no direct functional evidence is provided for any of these pathways.*

      __Answer: __We thank the reviewer for this thoughtful observation and agree that several interpretations linking phosphorylation changes to metabolic regulation, oxidative stress, proteostasis, and cellular quiescence are necessarily inferential in the absence of direct functional assays. With this in mind, we have revised the Discussion to more clearly distinguish data-driven observations from hypothesis-generating interpretations and have tempered language accordingly. These models are now explicitly framed as conceptual frameworks arising from large-scale phosphoproteomic analysis, intended to guide future targeted investigation rather than to assert definitive mechanistic conclusions (please see lines 588, 590, 596 - 599, 609, 613, 621 - 625 ).

      Given the breadth and depth of the dataset, only a limited number of functional pathways could be explored experimentally within the scope of the current study. We anticipate that the phosphoproteomic resource generated here, supported by the accompanying ShinySpermPhospho application, will enable the wider community to interrogate additional pathways and to design focused mechanistic studies building on these findings.

      • Acrosome reaction evaluation*

      I have encountered significant deficiencies in this approach. On one side, testing the effect of a single dose of inhibitors on a specific readout is too preliminary, as stated above. In addition, and due to the presence of possible off target effects, more than one inhibitor is expected to be tested, or a direct biochemical assay to confirm at least targeted action. Even KO models, as proposed for other proteins in Figure 7.

      Acrosome reaction values are expected to be presented, as regularly done, by indicating acrosome reacted percentages, without normalizations that complicate understanding. In addition, consider Pg as a more physiological stimulus instead of A23187 for triggering AR.

      __Answer: __We thank the reviewer for these constructive comments and agree that careful assessment of inhibitor effects on sperm viability and motility is essential. We would like to clarify, in case this was overlooked, that these controls were performed and are presented in Supplementary Figure S4. Specifically, sperm were exposed to four concentrations of each inhibitor and assessed over time (0 and 60 minutes) for viability, total motility, and progressive motility. Across all concentrations, time points, and treatment conditions, including combined inhibitor treatments, no significant reductions in sperm viability or motility parameters were observed. These data support the conclusion that the effects on acrosome reaction are not secondary to general sperm toxicity.

      With respect to data presentation, acrosome reaction values were expressed relative to matched capacitated vehicle-treated controls to account for biological variability in absolute acrosome reaction rates observed between independent sperm preparations and experimental days. This normalisation strategy was used to facilitate direct comparison between treatment groups, and respectfully, we have elected to retain this presentation format in Figure 6. Nonetheless, in the interest of transparency, we have now included the raw acrosome reaction values/ranges in the supplementary material (Table S6) and have provide these as a figure to the reviewer for reference.

      We agree that the use of progesterone represents a more physiological stimulus for inducing the acrosome reaction. However, there is no single universally accepted approach for acrosome reaction induction, and calcium ionophore-based assays remain widely used to assess the capacity for acrosomal exocytosis under defined experimental conditions. In the present study, this approach was selected to provide a robust and reproducible functional readout suitable for comparative analysis.

      We have revised the manuscript to more clearly describe the dose-response and viability control experiments, to acknowledge potential off-target effects of kinase inhibitors, and framed the acrosome reaction assays as functional screening experiments rather than definitive mechanistic dissection (please see lines 415, 705 - 710, Table S7).

      • *

      • Figure legend to Figure 7. How many oocytes, how many replicates were performed. How many transfers. Please add important data to the legend.*

      __Answer: __We thank the reviewer for highlighting this omission. In line with comment 4 from Reviewer #1 (please see above), we have revised the Figure 7 legend and Methods section to provide detailed information regarding sample size and experimental design, including the number of oocytes used per IVF experiment performed and the number of biological replicates.

      Specifically, IVF and oocyte isolation procedures were conducted according to standardised INFRAFRONTIER protocols (PMID: 25414328, 27262858, 38839949). Across knockout lines, IVF experiments were performed over 1 - 6 independent cycles per line, with an average of 24.2 oocytes used per cycle. To provide transparency regarding this variability, we have included a new supplementary figure (Figure S6) summarising the average number of oocytes used per IVF cycle alongside the corresponding cleavage rates (%CR).

      Sperm samples used in these assays were derived from cryopreserved cauda epididymal spermatozoa pooled from 10 heterozygous males per knockout line, as per EMMA guidelines (PMID: 17709347). Additionally , where available, we have incorporated reproductive outcome measures at the level of individual litters (e.g. average pup number and sex distribution) to provide further biological context. These additions improve transparency and ensure that the experimental design and data interpretation are clearly defined (please see lines 453 - 457, 1228 - 1234, 1431 - 1444, 1533 - 1536, 1572 - 1575, Figure 7 & S6).

      • *

      This is technically sophisticated phosphoproteomic study of mouse sperm maturation across the epididymis and during capacitation. The dataset is deep (>14,000 phosphosites) and the analyses integrate high-resolution MS, immunofluorescence, IPA, kinase mapping, pharmacological inhibition, and knockout mouse models.

      __Answer: __We thank the reviewer for this positive assessment and for recognising the technical sophistication and integrative nature of the study. We are also grateful for the constructive feedback provided, which has helped us to substantially strengthen the clarity, rigor, and presentation of the manuscript.

      PeerReviewed
    2. EMBOpress 20 May 2026

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

      Learn more at Review Commons

      Referee #3

      Evidence, reproducibility and clarity

      This is technically sophisticated phosphoproteomic study of mouse sperm maturation across the epididymis and during capacitation. The dataset is deep (>14,000 phosphosites) and the analyses integrate high-resolution MS, immunofluorescence, IPA, kinase mapping, pharmacological inhibition, and knockout mouse models. The manuscript represents a nice resource for the field. However, several issues limit clarity, mechanistic interpretation, and robustness of the conclusions. In particular, the manuscript's scope is extremely large, making some conclusions insufficiently supported, and some analyses require better control, methodological transparency, or deeper mechanistic connection. It gives the impression that some mechanistic data was added to descriptive data in order to increase the manuscript's impact, although the current mechanistic data is not convincing.

      Major concerns

      1. Conceptual Overreach - "Epididymal maturation accounts for 86% of phosphorylation changes" The manuscript repeatedly emphasizes that epididymal maturation causes the majority of phosphoproteomic remodeling. While the data indeed show large quantitative differences, several conceptual issues remain:
        • The caput vs. cauda comparison includes differences in protein abundance, not only phosphorylation.
        • Many phosphosites lost in the cauda may reflect protein loss, not dephosphorylation (the authors acknowledge this, but quantitative controls are insufficient).
        • The normalization method for phosphopeptide abundance vs total protein abundance is needed.
        • It is unclear whether the same amount of starting material and equal protein loading were used across stages.

      I wwould suggest to perform (or explicitly describe) normalization using matched proteome intensities. Provide supplementary plots showing phosphosite/parent-protein normalization to avoid overinterpreting phosphosite loss as dephosphorylation. 2. Capacitation analysis is underpowered and oversimplified The authors state that capacitation leads to "modest" changes. However: - The capacitation protocol uses dibutyryl-cAMP + pentoxifylline, which may bypass early physiological signaling. This is a important red flag 3. Kinase prediction and functional validation require more rigor The identification of 343 kinases that may regulate phospho-changes is extremely broad. Issues: - The kinase-substrate assignments rely heavily on in silico predictions (IPA, PhosphoSitePlus), which often contain non-sperm data. - Please explain the rationale by which, from 343 candidate kinases, 3 (STK33, HIPK4, PAK1) are selected. - The pharmacological inhibitors used have off-target effects (ML281 inhibits multiple CMGC kinases; Foretinib inhibits MET/VEGFR; NVS-PAK1-1 inhibits PAK1/2/3). - No control experiments are included to confirm kinase inhibition in sperm (e.g., phosphosite-specific Western blots). 4. The knockout-mouse validation section is underdeveloped The linkage of KO phenotypes to phosphorylation changes is potentially powerful but currently weak. Issues: - Most KOs are systemic deletions, not sperm-specific; phenotypes could stem from developmental defects. - Some proteins validated (e.g., ACO2, CMPK1) regulate core metabolism; their phenotypes may not reflect phosphoregulation but loss of essential protein function. - No evidence is provided that the KO affects the specific phosphosites detected in the MS dataset. 5. Immunofluorescence and Western blots need improved quantification Figures showing PKA substrate, pY, pT, pS changes are visually compelling but lack: - quantification across biological replicates - explanation of antibody specificity (e.g., pan-PKA sites include RRXS/T motifs; cross-reactivity possible). 6. Many interpretations about metabolism, storage, oxidative stress, and quiescence are speculative The discussion provides attractive models linking phosphorylation to: - suppression of glycolysis - quiescent metabolic state in cauda epididymis - activation of antioxidant pathways - UPR and proteostasis modifications However, no direct functional evidence is provided for any of these pathways. 7. Acrosome reaction evaluation I have encountered significant deficiencies in this approach. On one side, testing the effect of a single dose of inhibitors on a specific readout is too preliminary, as stated above. In addition, and due to the presence of possible off target effects, more than one inhibitor is expected to be tested, or a direct biochemical assay to confirm at least targeted action. Even KO models, as proposed for other proteins in Figure 7. Acrosome reaction values are expected to be presented, as regularly done, by indicating acrosome reacted percentages, without normalizations that complicate understanding. In addition, consider Pg as a more physiological stimulus instead of A23187 for triggering AR. 8. Figure legend to Figure 7. How many oocytes, how many replicates were performed. How many transfers. Please add important data to the legend.

      Significance

      This is technically sophisticated phosphoproteomic study of mouse sperm maturation across the epididymis and during capacitation. The dataset is deep (>14,000 phosphosites) and the analyses integrate high-resolution MS, immunofluorescence, IPA, kinase mapping, pharmacological inhibition, and knockout mouse models.

      PeerReviewed
    3. EMBOpress 20 May 2026

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

      Learn more at Review Commons

      Referee #2

      Evidence, reproducibility and clarity

      Summary:

      In this manuscript, the authors examine dynamic modifications of the sperm phosphoproteome during epididymal transit and capacitation. They compare three distinct populations differing in anatomical localization and activation status: caput sperm, non‑capacitated cauda sperm, and capacitated cauda sperm. Using high‑resolution tandem mass spectrometry, they reveal that phosphorylation changes during epididymal passage are far more extensive than previously appreciated. These findings are further validated in genetically modified animal models, where disruption of selected genes encoding for phosphoproteins results in marked defects in sperm motility and fertilization capacity.

      Major points:

      Throughout the text, and particularly in the paragraph entitled 'Epididymal maturation accounts for the majority of maturation‑associated sperm cell signaling,' it seems that phosphorylation is interpreted as inherently activatory and dephosphorylation as inhibitory (lines 248-252). Since this relationship is not universally applicable, it would be valuable to address this issue at the outset of the paragraph and to discuss how phosphorylation events are context‑dependent in their effects on protein function.

      Lines 392-393: the claim that "the introduction of each inhibitor to populations of capacitating spermatozoa led to a significant reduction..." is not fully supported by data and should be toned down. In fact, two out of three inhibitors, do not significantly affect the acrosome reaction.

      The discussion section, spanning 11 pages, is overly long and contains considerable repetition. I recommend transferring the detailed description of experiments to the 'Results' section and using the discussion primarily to synthesize and highlight the novel findings while limiting speculative content. For example, the content in lines 509-530 could be condensed and relocated to the Results. Likewise, other detailed examples would be more appropriately presented within their respective result paragraphs.

      Minor points:

      • To improve reproducibility, the suppliers of all reagents should be specified together with their catalogue numbers.
      • Figure 7: it is unclear which data are statistically significant
      • Figure 7B: fertilization capacity should be assessed at an earlier stage, as the cleavage rate to 2-cell embryo may be affected by factors unrelated to the sperm ability to fertilize

      Significance

      A key novel finding of this work is that extensive changes in the sperm phosphoproteome occur during epididymal maturation, whereas capacitation is associated with comparatively modest modifications. This research provides a finely resolved description of phosphorylation events associated with the signaling pathways underlying functional sperm maturation. The methodological innovation -high‑resolution MS‑based phosphoproteomics- unlocks a level of detail and comprehensiveness in phosphorylation analysis that was previously unattainable. Moreover, the identification of previously unrecognized phosphoproteins in sperm cells, together with the development of a dedicated application hosting the complete dataset, represents a valuable resource for researchers in reproductive biology and particularly for experts in sperm development and maturation.

      PeerReviewed
    4. EMBOpress 20 May 2026

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

      Learn more at Review Commons

      Referee #1

      Evidence, reproducibility and clarity

      The manuscript by Skerrett-Byrne and collaborators represents a comprehensive and technically sophisticated phosphoproteomics study. Using high-resolution mass spectrometry on mouse sperm obtained from the caput and cauda regions of the epididymis, both before and after capacitation, the authors generated a more complete database of phosphorylation changes in these cells. One of the most interesting outcomes is that most of these changes occur during sperm maturation, rather than sperm capacitation. The work is important and relevant, and the information obtained could be valuable for reproductive biologists working in basic science, as well as for the identification of novel contraceptive targets.

      Minor comments:

      1) The title should include a reference to sperm capacitation, as most of the study focuses on comparisons between epididymal maturation and capacitation, and the functional experiments are based on the latter.

      2) Considering the newly reported changes in phosphosites, it would be desirable to include validation at the individual protein level for at least a few examples, using an independent technique such as western blotting.

      3) In the knockout models, it is not possible to distinguish between defects in spermatogenesis and those arising during maturation or capacitation. A parameter directly related to spermatogenesis should therefore be included, for example, testicular weight or histology, sperm number, and sperm morphology.

      4) Error values, sample size, and statistical analyses are missing from Figure 7 and should be provided for clarity.

      Significance

      This study shows by high-resolution phosphoproteomics that most phosphorylation changes occur during epididymal transit rather than capacitation, challenging long-standing assumptions. The integration of the new datasets with functional validation of key kinases and knockout models strengthens the study; however, the work lacks single-protein validation of phosphorylation events, and the use of systemic knockouts does not allow confirmation of sperm-specific effects. The open ShinySpermPhospho dataset will be worthwhile to a broad audience of reproductive biologists and cell signaling specialists, and may be of value for future studies on male fertility and the development of novel male contraceptives.

      Field of expertise: reproductive physiology, sperm biology and capacitation, gamete interaction.

      PeerReviewed
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      Que até os protocolos de recuperação possuem respondentes e não respondentes dependendo de cada intervenção

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      Start at 00 → 02 to understand the platform shell and how a project is created. Then read each Hub in pipeline order (03 → 10). Finally, read the cross-cutting docs (11 → 14) which apply across every hub.

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