I like that each of the center buttons open in a new window, but opening in a new window without flagging that is getting us dinged in Silktide. Do we need to add an "open in new window" icon next to the text on anything that will do that?

Two typos in the CEWIT alt text.

These should all be H2s.

The image on this features Far Beyond banners. We will need an updated image.

I wonder if there is any way a company is able to store secure information in a way such that even despite a breach in security, the information is still inaccessible to hackers. Would it be possible for the information to be stored instead on the users platform, and whenever the server needs to access it, it will instead request from the users computer? While this is less convenient and I assume it would require more processing time, it would be more secure as all information is not centralized.

I find it interesting that this article was released in 2021, but the incidents cited took place in 2015. Does this mean these incidents in which Facebook employees abused their power were not open to the public for 6 years?

I find it shocking that the personal information that you willingly give to social media platforms can be so easily stolen and posted publicly on the internet. That is a huge breach of trust as people put their trust into these companies to save and protect their personal information.

I think the sentence "Although we are concerned about information privacy, we often share information with social media platforms and believe they will safely keep these details" is particularly true. We always say we want to protect privacy, but in reality, we still readily click "agree" and hand over our information without hesitation. Seeing this sentence made me reflect a bit - perhaps we have become too accustomed to convenience, and thus have overlooked the aspect of security.

This post is very interesting because it does not technically have any means for a lawsuit. If celebrities who could have gotten their information leaked, that could ruin their phones with the number of people who try to contact them after their information is leaked.

This section makes me think on the internet nowadays, there's absolutely no way to keep your information to yourself. People's information is in so many different companies, and users would not know how their information is being used either. Users has no control over their own privacy although it's something about themselves.

The movie Catch Me if You Can is a fantastic movie and personally I think it's Leonardo DiCaprio's best movie along with the Wolf on Wall Street. In this movie, he is able to complete one of the largest bank fraud heists ever by becoming a 'pilot'. He does it by falsely becoming a pilot and then forging checks to his name. Something like this would never be able to happen in today's age, there's just too much security and restrictions when it comes to money and taxes. I think the best part of the whole movie is at the end when instead of being sentenced to prison for life, he is able to work with the FBI and help catch other criminals who did the same thing as him.

This is an article about how Facebook employee abusing their access of data to harass women. The author of the book An Ugly Truth: Inside Facebook’s Battle for Domination wrote about the crises that happened within the Facebook company over the past five years. It is very scary to me how employee abuses their power and put women in such dangerous position; and the woman won’t be able to know how her information gets leaked.

This article outlines the potential hazards and dangers of parents sharing photos and content of their children before they are old enough to consent. I believe this issue has only become more relevant since this article was published in 2020, with the rise of "family channels" and family-related content online. Parents are going beyond just sharing pictures; they are forcing their children into staged content, making them constantly perform the occurrences of their daily lives.

I’m not surprised that large companies like Facebook are mishandling their users’ private information. What’s really troubling is that even when such companies are exposed, or even sued, the actual victims rarely receive meaningful compensation. It feels deeply unfair.

Sometime before August 2019, Facebook's database was breached and the personal information of 530 million people was stolen and made public. I find it shocking that the company can decide not to notify their users that their information was stolen.

This article is documenting that Facebook stored passwords of users in a way that made them accessible to around 200,000 employees. I don't fear my information being "leaked" due to lack of assets that are desirable but that could just be a lack of understanding but this did concern me.

This source discusses the right to privacy and each person's right to privacy. It discusses the law and the United States rights that make sure that each citizen has the right to have their own personal and private information and lives.

I read this report from NPR titled "After Data Breach Exposes 530 Million, Facebook Says It Will Not Notify Users". I was really shocked. The data of 53 million people was leaked, and yet the company chose not to notify the users? This made me realize that big companies claim to "value privacy", but when problems occur, their first reaction is usually to protect themselves rather than protect the users. After reading it, I will be more cautious about the personal information on social media. After all, sometimes "the sense of security" is just an illusion.

Last Reviewed 10/19/2024 (v2.4 Update)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Urothelial Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

v2.4 Update Summary

• The FDA granted accelerated approval of enfortumab vedotin in combination with pembrolizumab for the treatment of adult patients with locally advanced or metastatic urothelial cancer in December 2023 [Ref 3, 158].

• The FDA granted approval of nivolumab in combination with cisplatin and gemcitabine as first-line treatment of adult patients with unresectable or metastatic urothelial carcinoma in March 2024 [Ref 6].

• Data have been reported indicating similar responses to ICI therapy regardless of FGFR3 mutation status for patients with metastatic urothelial cancer [Ref 159].

• The FDA granted approval of nogapendekin alfa inbakicept with BCG for adult patients with BCG-unreseponsive NMIBC with carcinoma in situ with or without papillary tumors in April 2024 [Ref 160].

v2.3 Update Summary

• Atezoluzimab for the treatment of cisplatin- and platinum-ineligible patients with mUC was voluntary withdrawn by the manufacturer in November, 2022 [Ref 161].

• The FDA granted approval of nadofaragene firadenovec for the treatment of BCG-unresponsive NMIBC with CIS with or without papillary tumors in December 2022 [Ref 162].

• The FDA granted accelerated approval of enfortumab vedotin with pembrolizumab for the treatment of patients with locally advanced or metastatic urothelial carcinoma who are ineligible for cisplatin-containing chemotherapy in April 2023 [Ref 3, 158].

v2.2 Update Summary * The indication of pembrolizumab for the treatment of patients with mUC ineligible for platinum-containing chemotherapy or with disease progression on or after platinum-containing chemotherapy was revised in August 2021 to no longer specify PD-L1 status for eligibility [Ref 3].

v2.1 Update Summary * The FDA granted approval of nivolumab for the treatment of patients with urothelial cancer who are at high risk of recurrence after undergoing radical resection in August 2021 [Ref 6].

See the highlighted text for updated content and more detailed information.

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Urothelial Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

Update 8-17-22

Based on advances in the field, including the following three FDA approvals and ongoing trials, the RCC CPG has been updated throughout the entire manuscript:

(1) Pembrolizumab for the adjuvant treatment of RCC: In November of 2021, the FDA approved pembrolizumab (anti-PD-1) for the adjuvant treatment of resected RCC at intermediate-high or high risk of recurrence following nephrectomy, or following nephrectomy and resection of metastatic lesions.

Reference: FDA approves pembrolizumab for adjuvant treatment of renal cell carcinoma FDA press release

(2) Pembrolizumab plus lenvatinib for the treatment of advanced RCC: In August of 2021, the FDA approved pembrolizumab in combination with lenvatinib (a VEGF receptor tyrosine kinase inhibitor; TKI) for the first-line treatment of patients with advanced RCC (aRCC).

Reference: FDA approves lenvatinib plus pembrolizumab for advanced renal cell carcinoma FDA press release

(3) Nivolumab plus cabozantinib for the treatment of advanced RCC: In January of 2021, the FDA approved nivolumab (anti-PD-1) in combination with cabozantinib (a VEGF receptor TKI) for the first-line treatment of patients with aRCC.

Reference: FDA approves nivolumab plus cabozantinib for advanced renal cell carcinoma FDA press release

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Prostate Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

Update August 2022

Based on the tissue agnostic approvals for pembrolizumab for the treatment of TMB-H and MSI-H/dMMR solid tumors and dostarlimab for the treatment of dMMR solid tumors, and their approved companion diagnostics, the Prostate Cancer CPG has been updated in the following location: - Introduction

References: Pembrolizumab (KEYTRUDA) TMB-H tissue-agnostic approval

Pembrolizumab (KEYTRUDA) MSI-H/dMMR tissue-agnostic approval

Dostarlimab (JEMPERLI) dMMR tissue-agnostic approval

Update 8-24-22

Based on new approvals of immunotherapy agents for the treatment of prostate cancer and new data that has been published since its original publication, the Prostate CPG was updated in the following locations: - Figure 1 - Prostate Cancer Treatment Algorithm

v1.1 Update

Since guideline publication, this phase II study assessing neoadjuvant cemiplimab reported a pCR rate of 51% and a major pathologic response rate of 13% in 70 patients with resectable stage II, III, or IV (M0) CSCC. Notably, an additional 9 patients were treated but did not undergo surgery. (This regimen was not FDA-approved at the time of update v1.1). [Ref 179]

v1.1 Update

In addition to the FDA-approved ICIs described in this section, since guideline publication, neoadjuvant anti-PD-1 therapy prior to curative-intent surgery demonstrated efficacy in a phase II study. (This regimen was not FDA-approved at the time of update v1.1.) [Ref 179]

v1.1 Update

In addition to the approved ICIs for MCC described in this section, since guideline publication retifanlimab was granted accelerated approval by the FDA in March 2023 for the treatment of metastatic or recurrent locally advanced MCC. Approval was based on the POD1UM-201 trial (NCT03599713). The primary outcome measures for approval were ORR and DOR (Table 2). Safety was assessed in 105 patients with MCC, where retifanlimab was determined to be safe and well-tolerated. Serious AEs occurred in 22% of patients, and the most common serious AEs were fatigue, arrhythmia, and pneumonitis. Permanent discontinuation of therapy due to AEs occurred in 11% of patients. [Ref 177, 178]

Additionally, although not FDA-approved at the time of update v1.1, two studies combining an anti-PD-(L)1 ICI with an anti-CTLA-4 ICI have reported efficacy in patients with recurrent/metastatic MCC. [Ref 180, 181]

Last reviewed 3/19/2024 (v1.1 Update)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication and made with the approval of the SITC NMSC CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.1 Update Summary

• The FDA granted accelerated approval for retifanlimab (anti-PD-1 ICI) for the treatment of adult patients with metastatic or recurrent locally advanced MCC in March 2023. The NMSC CPG was updated in the following locations: Introduction, Merkel Cell Carcinoma, Recommended Immunotherapies for MCC, Figure 1 – FDA-Approved ICI agents for NMSC, Table 2 – NMSC Landmark Clinical Trial Data Leading to FDA Approvals for ICIs for MCC and Novel Strategies and Promising Future Directions. [Ref 177, 178]

• Data have been reported demonstrating efficacy with neoadjuvant anti-PD-1 ICI therapy prior to curative-intent surgery in patients with CSCC. Based on these practice-changing data, the NMSC CPG was updated in the following locations: Based on these practice-changing data, the NMSC CPG was updated in the following locations: Recommended Immunotherapies for CSCC, and Novel Strategies and Promising Future Directions for CSCC. [Ref 179]

• Data have been reported demonstrating efficacy combining an anti-PD-(L)1 ICI with an anti-CTLA-4 ICI for patients with advanced MCC. Based on these practice-changing data, the NMSC CPG was updated in the following locations: Merkel Cell Carcinoma, Recommended Immunotherapies for MCC, and Novel Strategies and Future Directions for CSCC. [Ref 180, 181]

See highlighted text for updated content and more detailed information.

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Multiple Myeloma Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

Update 2-1-2022

Based on the FDA approval of daratumumab in combination with carfilzomib and dexamethasone for adult patients with relapsed or refractory multiple myeloma who have received 1 to 3 prior lines of therapy on November 30, 2021, the Multiple Myeloma CPG has been updated in the following locations: * Monoclonal Antibodies Expert Panel Recommendations * Multiple Myeloma Key Monoclonal Antibody Therapies Trials

Reference: Daratumumab + hyaluronidase-fihj (Darzalex Faspro, Janssen Biotech, Inc.) and carfilzomib (Kyprolis, Amgen, Inc.) plus dexamethasone press release

Update 4-9-2021

Based on the approval of idecabtagene vicleucel for the treatment of adult patients with relapsed or refractory multiple myeloma after four or more prior lines of therapy, including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody, the Multiple Myeloma CPG has been updated in the following locations: * Background * CAR T cell Therapies

Reference: Idecabtagene vicleucel (ABECMA) FDA press release

Based on the approval of isatuximab-irfc in combination with carfilzomib and dexamethasone, for the treatment of adult patients with relapsed or refractory multiple myeloma who have received one to three prior lines of therapy, the Multiple Myeloma CPG has been updated in the following locations: * Monoclonal Antibody Therapies * Multiple Myeloma Key Monoclonal Antibody Therapies Trials

Reference: Isatuximab-irfc (SARCLISA) FDA press release

Update 8-5-2020

Based on the accelerated approval of belantamab mafodotin-blmf for adult patients with relapsed or refractory multiple myeloma who have received at least 4 prior therapies, including an anti-CD38 monoclonal antibody, a proteasome inhibitor, and an immunomodulatory agent, the Multiple Myeloma CPG was updated in the following locations: * Antibody-Drug Conjugates * Background

Reference: Belantamab mafodotin-blmf (BLENREP) FDA press release

Last reviewed 02/10/2025 (v1.4 Update)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Lymphoma Cancer Immunotherapy Guideline Expert Panel. More information on the SITC Guidelines can be found at sitcancer.org/guidelines.

v1.4 Update Summary

• The FDA granted approval of denileukin diftitox-cxdl for the treatment of patients with stage 1 to 3 relapsed/refractory cutaneous TCL after at least 1 prior systemic therapy in August 2024 [Ref 242-243].

• The FDA granted accelerated approval of epcoritamab for adult patients with relapsed or refractory FL after two or more lines of systemic therapy in June 2024 [Ref 244, 245].

• The FDA granted approval of lisocabtagene maraleucel for adult patients with relapsed or refractory mantle cell lymphoma MCL who have received at least two prior lines of systemic therapy, including a Bruton tyrosine kinase inhibitor (BTKi) in May 2024 [Ref 246].

• The FDA granted accelerated approval of lisocabtagene maraleucel for adults with relapsed or refractory FL who have received two or more prior lines of systemic therapy in May 2024 [Ref 246].

• The FDA granted accelerated approval of lisocabtagene maraleucel for adult patients with relapsed or refractory CLL or SLL who have received at least 2 prior lines of therapy, including a BTKi and a B-cell lymphoma 2 (BCL-2) inhibitor in March 2024 [Ref 246].

• The FDA granted accelerated approval of zanubrutinib with obinutuzumab for relapsed or refractory FL after two or more lines of systemic therapy in March 2024 [Ref 247, 248].

• The FDA granted accelerated approval of glofitamab for relapsed or refractory diffuse large B-cell lymphoma DLBCL, not otherwise specified (NOS) or LBCL arising from FL, after two or more lines of systemic therapy in June 2023 [Ref 249].

• The FDA granted accelerated approval of epcoritamab for relapsed or refractory DLBCL NOS, including DLBCL arising from indolent lymphoma, and high-grade B-cell lymphoma (HGBL) after two or more lines of systemic therapy in May 2023 [Ref 244].

• The FDA granted approval of polatuzumab vedotin with a rituximab product, cyclophosphamide, doxorubicin, and prednisone (R-CHP) for adult patients who have previously untreated DLBCL, NOS or HGBL and who have an International Prognostic Index (IPI) score of 2 or greater in April 2023 [Ref 250, 251].

• The FDA granted accelerated approval of mosunetuzumab for adult patients with relapsed or refractory FL after two or more lines of systemic therapy in December 2022 [Ref 252].

• Data have been reported on the safety and efficacy of lenalidomide + obinutuzumab for relapsed or refractory FL and marginal zone lymphoma [Ref 253, 254].

• Data have been reported on the safety and efficacy of nivolumab + AVD for patients with Hodgkin lymphoma [Ref 255].

• Data have been reported on the safety and efficacy of nivolumab + chemotherapy and pembrolizumab + chemotherapy for patients with relapsed or refractory Hodgkin lymphoma [Ref 256–258].

• Data have been reported on the safety and efficacy of BV-nivolumab for the treatment of R/R PMBCL [Ref 259].

v1.3 Update Summary

• The FDA granted approval of lisocabtagene maraleucel for the treatment of adult patients with LBCL who have refractory disease to first-line chemoimmunotherapy or relapse within 12 months of first-line chemoimmunotherapy; or refractory disease to first-line chemoimmunotherapy or relapse after first-line chemoimmunotherapy and are not eligible for hematopoietic stem cell transplantation (HSCT) due to comorbidities or age in June 2022 [Ref 246].

• The FDA granted accelerated approval of tisagenlecleucel for the treatment of adult patients with relapsed or refractory FL following 2 or more lines of systemic therapy in May 2022 [Ref 113].

• The FDA granted approval of axicabtagene ciloleucel for adult patients with LBCL that is refractory to first-line chemoimmunotherapy or relapses within 12 months of first-line chemoimmunotherapy in April 2022 [Ref 111].

• The FDA granted accelerated approval of axicabtagene ciloleucel for adult patients with relapsed or refractory FL after two or more lines of systemic therapy in March 2021 [Ref 111].

v1.2 Update Summary

• The FDA granted accelerated approval of loncastuximab tesirine-lpyl for the treatment of adult patients with relapsed or refractory LBCL after two or more lines of systemic therapy, including DLBCL NOS, DLBCL arising from low grade lymphoma, and HGBL in April 2021 [Ref 260].**

v1.1 Update Summary

• The FDA granted approval of lisocabtagene maraleucel for the treatment of R/R large B-cell lymphoma after two or more lines of systemic therapy, including DLBCL not otherwise specified (including DLBCL arising from indolent lymphoma), high-grade B-cell lymphoma, PMBL, and FL grade 3B in February 2021. [Ref 246, 261]

Last reviewed 6/16/23 (v1.1(A) update supplement)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC HCC CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

A supplementary immunotherapy treatment algorithm for HCC was generated based on the Expert Panel recommendations. It can be found on the SITC website here.

Last reviewed 6/16/23 (v1.1(A) update supplement)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC HCC CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

SITC published an addendum to the guideline (Addendum 1, update v1.1(A)) based on the approval of tremelimumab in combination with durvalumab for adult patients with unresectable HCC. A supplementary immunotherapy treatment algorithm for HCC was generated based on the Expert Panel recommendations. It can be found on the SITC website here.

Last Reviewed 11/15/2024 (v1.1 Update)

The information on this page provides a detailed overview of updates to the guideline content based on changes in the field. Updates to the guideline outlined below were made with the approval of SITC's Head and Neck Squamous Cell Carcinoma (HNSCC) Guideline Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

Update v1.1 Summary * The FDA approved pembrolizumab for patients with recurrent or metastatic cutaneous squamous cell carcinoma in June 2020 [Ref 108, 169].

• The FDA approved toripalimab in combination with cisplatin and gemcitabine for first-line treatment of patients with metastatic or recurrent locally advanced nasopharyngeal carcinoma, or as a monotherapy for treatment of adult patients with recurrent, unresectable, or metastatic nasopharyngeal carcinoma with disease progression on or after platinum-containing chemotherapy in October 2023 [Ref 170, 171].

• Practice-changing data have been reported from CheckMate 141 regarding nivolumab as a first-line treatment in recurrent or metastatic HNSCC after progressing on platinum therapy for locally advanced disease in the adjuvant or primary (ie, with radiation) setting [Ref 172].

• Practice-changing data have been reported from KEYNOTE B10 and the FRAIL-IMMUNE/GORTEC 2018-03 trials, demonstrating efficacy of combining an anti-PD-(L)1 immune checkpoint inhibitor (ICI) with carboplatin + paclitaxel in frail patients with R/M HNSCC [Ref 173, 174].

Last Reviewed 5/25/24 (v1.1 Update)

The information on this page provides a detailed overview of updates to the guideline content based on changes in the field. Updates to the guideline outlined below were made with the approval of SITC's Gynecology Cancer CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.1 Update Summary

• The FDA granted approval of pembrolizumab with chemoradiotherapy FIGO 2014 Stage III-IVA cervical cancer on January 12, 2024. Based on this approval, the Gynecologic Cancer CPG has been updated in the following location: Immunotherapy for the treatment of cervical cancer - Recommended immunotherapy treatments for cervical cancer (including Table 2). [Ref 13, 299, 300]

• The FDA granted approval of dostarlimab with carboplatin and paclitaxel, followed by single-agent dostarlimab for dMMR or MSI-H (as determined by an FDA-approved test) primary advanced or recurrent endometrial cancer on July 31, 2023. Based on this approval, the Gynecologic Cancer CPG has been updated in the following location: Immunotherapy for the treatment of endometrial cancer - Recommended immunotherapy treatments for endometrial cancer (including Table 3). [Ref 15, 121, 301]

See highlighted text for updated content and more detailed information.

Last reviewed 5/28/2025 (v1.1 Update)

Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication are made with the approval of the SITC Gastrointestinal Cancer CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.1 Update Summary

• The FDA approved nivolumab in combination with ipilimumab for adult and pediatric patients 12 years of age and older with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) colorectal cancer (CRC) in April 2025 [Ref 242, Ref 243].

• The FDA approved tislelizumab in combination with platinum and fluoropyrimidine-based chemotherapy for the first-line treatment of adult patients with unresectable or metastatic HER2-negative gastric or gastroesophageal junction adenocarcinoma whose tumors express PD-L1 (≥1) in December 2024 [Ref 229].

• The FDA approved zolbetuximab-clab in combination with platinum and fluoropyrimidine-containing chemotherapy for the first-line treatment of adults with locally advanced unresectable or metastatic HER2-negative gastric or gastroesophageal junction adenocarcinoma whose tumors are claudin (CLDN) 18.2 positive as determined by an FDA-approved test in October 2024 [Ref 230].

• The FDA approved tislelizumab for the treatment of adult patients with unresectable or metastatic esophageal squamous cell carcinoma (ESCC) after prior systemic chemotherapy that did not include a PD-L1 inhibitor in March 2024 [Ref 229].

• The indication of pembrolizumab with trastuzumab, fluoropyrimidine, and platinum-containing chemotherapy for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma was restricted to patients with CPS greater than or equal to 1, as determined by an FDA-approved test, in November 2023 [Ref 231].

• The FDA approved pembrolizumab with fluoropyrimidine- and platinum-containing chemotherapy as first-line treatment for adults with locally advanced unresectable or metastatic HER2-negative gastric or gastroesophageal junction (GEJ) adenocarcinoma in November 2023 [Ref 231].

• The FDA approved pembrolizumab with gemcitabine and cisplatin for the treatment of locally advanced unresectable or metastatic biliary tract cancer (BTC) in October 2023 [Ref 231].

• Additional changes made to address practice-changing data and updates in the field.

See the highlighted text for updated content and more detailed information.

v1.1 Update

Since guideline publication, additional neoadjuvant/pre-operative studies have shown efficacy with ICIs for patients with dMMR/MSI-H gastric/GEJ adenocarcinoma. These regimens were not FDA approved at the time of update v1.1 [Ref 96, 97, 160, 232, 233].

Last Reviewed 3/15/2024 (v1.2 Update)

The information on this page provides a detailed overview of updates to the guideline content based on changes in the field. Updates to the guideline outlined below were made with the approval of SITC's Breast Cancer CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.2 Update Summary

• The FDA granted accelerated approval for dostarlimab for the treatment of dMMR recurrent or advanced solid tumors (along with the VENTANA MMR RxDx Panel companion diagnostic to detect dMMR) that have progressed on or following prior treatment and who have no satisfactory alternative treatment options on August 17, 2021. Based on these approvals, the Breast Cancer CPG has been updated in the following locations: Immunotherapy with PD-(L)1 Inhibitors for the Treatment of Advanced/Metastatic Breast Cancer and Diagnostics and Biomarker Testing in Patients with Advanced/Metastatic Breast Cancer. [Ref 290]

• The FoundationOne CDx assay was approved as the companion diagnostic for identifying patients with MSI-H tumors for treatment with pembrolizumab in February 2022. Based on this approval, the Breast Cancer CPG has been updated in the following locations: Diagnostics and Biomarker Testing in Patients with Advanced/Metastatic Breast Cancer. [Ref 291]

See highlighted text for updated content and more detailed information.

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

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Reply to the reviewers

Point-by-Point Response to Reviewers for Manuscript #RC-2024-02720

Manuscript Title: Molecular and Neural Circuit Mechanisms Underlying Sexual Experience-dependent Long-Term Memory in Drosophila.

Corresponding Author: Woo Jae Kim

We extend our sincere gratitude to the Managing Editor and both reviewers for their diligent and insightful evaluation of our manuscript. The comprehensive feedback provided has been invaluable, guiding us to significantly strengthen the manuscript's scientific rigor, logical cohesion, and overall impact. We have undertaken a substantial revision, incorporating new experimental evidence, reframing the central narrative, and improving data presentation to address all concerns raised.

The major revisions include:

1. New Experimental Evidence: We have performed three new sets of experiments to address key questions raised by the reviewers. First, we used the protein synthesis inhibitor cycloheximide to pharmacologically validate that the observed memory is indeed a form of long-term memory (LTM). Then, we performed genetic intersectional analyses to determine if the identified Yuelao (YL) neurons express the canonical sex-determination transcription factors doublesex (dsx) and fruitless (fru).
2. Narrative Reframing and Logical Restructuring: We fully agree with the reviewers that the logic of the original manuscript was confusing, particularly regarding the distinction between the broad Mushroom Body (MB) Kenyon Cell (KC) population and the specific YL neurons. The manuscript has been extensively rewritten to present a clear, hypothesis-driven narrative. We now frame the initial KC-related findings as part of a broader screening effort that logically led to the identification and focused investigation of the YL neuron circuit.
3. Refined Central Claim: Guided by the reviewers' feedback and our new data, we have sharpened our central claim. We now propose that YL neurons constitute a critical circuit for forming attractive taste- and pheromone-based memories derived from Gr5a neuronal inputs. This form of appetitive memory is distinct from the previously characterized internal reward state associated with ejaculation, adding a new layer to our understanding of how male flies remember and evaluate reproductive experiences.
4. Improved Data Quality and Analysis: In response to valid critiques, all imaging figures have been replaced with high-resolution versions. Furthermore, our methods for fluorescence quantification, particularly for the TRIC calcium imaging experiments, have been corrected to include normalization against an internal reference channel, adhering to established best practices. All requested genetic control experiments have been performed. We are confident that these comprehensive revisions have fully addressed all concerns and have transformed our manuscript into a much stronger, more focused, and logically sound contribution. We thank you again for the opportunity to improve our work and look forward to your evaluation of the revised manuscript.

Responses to Reviewer #1

General Comments: This study explores the molecular and neural circuitry mechanisms underlying sexual experience-dependent long-term memory (SELTM) in male Drosophila. The authors use behavioral, imaging, and bioinformatics approaches to identify YL neurons, a subset of mushroom body (MB) projecting neurons, as crucial for SELTM formation. They propose that YL neurons receive inputs from WG neurons via the sNPF-sNPFR pathway and implicate molecular players such as orb2, fmr1, MDAR2-CaMK, and synaptic plasticity in their function.

However, the evidence presented does not adequately support the authors' claims. The data fail to cohesively tell a logical story, and key conclusions appear to be based on assumptions and correlations rather than robust evidence.

• Answer: We are deeply grateful to both reviewers for their thorough and constructive evaluation of our manuscript. Their collective feedback has been instrumental in helping us to clarify the study's rationale, strengthen our interpretations, and significantly improve the overall quality and impact of the work. We appreciate the recognition of our study's potential to advance the understanding of how sexual experience modifies future mating behaviors and to elucidate the neuronal and molecular mechanisms of how memory regulates a key sexual behavior in male Drosophila*.

• *In response to the general comments, we have undertaken a major revision of the manuscript to improve the clarity, logic, and presentation. We have rewritten the Abstract and Introduction to more clearly define "sexual experience-dependent long-term memory" (SELTM) and articulate its significance in the context of adaptive decision-making and interval timing. The entire manuscript has been restructured to present a more logical, hypothesis-driven narrative that clearly distinguishes our initial broad screening from the focused investigation of the YL neuron circuit. We have also incorporated alternative interpretations of our data, particularly regarding the role of the YL circuit in regulating baseline mating duration in naive males, which has added more depth to the study. Finally, all figures have been remade in high resolution, and all requested genetic controls and methodological clarifications have been added to ensure rigor and reproducibility. We are confident that these revisions have addressed the reviewers' concerns and have resulted in a much stronger manuscript.

Comment 1: The study identifies the knowledge gap (lines 103-104) but fails to integrate relevant literature, particularly Shohat-Ophir et al., Science (2012), and Zer-Krispil et al., Curr Biol (2018). These studies established that ejaculation induces appetitive memory in male Drosophila via corazonin and NPF neurons. The current study does not provide direct evidence that the "act of mating itself" drives SELTM, as it includes both courtship and copulation.

Response: Thank you for highlighting these two landmark studies. We fully agree that Shohat-Ophir et al., Science (2012) and Zer-Krispil et al., Curr Biol (2018) were pivotal in demonstrating that ejaculation—and the accompanying corazonin/NPF signalling—can establish an appetitive memory in males.

In the revised manuscript we have now integrated both papers on lines 111-118:

“Previous work has shown that successful copulation is intrinsically rewarding to male Drosophila: a single mating encounter elevates brain neuropeptide F (NPF) levels and suppresses subsequent ethanol preference19. Importantly, Zer-Krispil et al. further demonstrated that ejaculation itself—artificially induced by optogenetic activation of corazonin (Crz) neurons—is sufficient to mimic this reward state, driving appetitive memory formation and up-regulation of NPF. These findings indicate that the act of ejaculation, rather than the entire courtship sequence, is the critical sensory event that gates post-mating reward.”

Comment 2: The nature of the observed long-lasting reduced mating duration requires clearer characterization: Is this an associative memory or experience-dependent behavioral plasticity? Can the formation of this long-term memory be blocked by protein synthesis inhibitors, such as cycloheximide?

Response: We thank the reviewer for this excellent suggestion to pharmacologically characterize the nature of the memory. To definitively test whether the observed SMD is a form of protein synthesis-dependent long-term memory (LTM), we performed a new experiment as suggested.

We have now included data in new Figure supplement 1I showing that feeding males the protein synthesis inhibitor cycloheximide (CXM) for 24 hours immediately following the sexual experience completely blocks the formation of the long-lasting SMD phenotype. Control flies fed a vehicle solution exhibited robust SMD. This result provides strong evidence that SELTM is not merely a form of transient behavioral plasticity but is a genuine form of LTM that requires de novo protein synthesis for its consolidation, a hallmark of LTM across species.[1]

The revised text was put on lines 173-176:

" To determine whether the persistent reduction in mating duration (SMD) depends on de-novo protein synthesis, we fed males the translational inhibitor cycloheximide (CXM). Under this regimen, CXM completely abolished the SMD phenotype (Fig. 1I)."

Comment 3: While schematics illustrate the working hypotheses, the text lacks detailed explanations, leaving the reader unclear about the rationale behind certain conclusions.

__Response: __Thank you very much for this insightful comment. We fully agree that the original manuscript did not provide sufficient textual justification for the conclusions derived from the schematics. In the revised version we have therefore added comprehensive explanations immediately following each figure (or schematic) that explicitly state the underlying rationale, the key observations supporting our hypotheses, and the logical steps leading to each conclusion. We believe these additions now make the reasoning transparent and easy to follow. We appreciate your feedback, which has substantially improved the clarity of our work.

• *

Comment 4*: The logic to draw certain conclusions was confusing and misleading. - For instance, the role of orb2 in SELTM is examined via knockdown in MB Kenyon cells (KCs) (using ok107>orb2-RNAi), which is irrelevant to the claim that orb2 functions in YL neurons. Additionally, RNAseq analyses (Fig. 1N-S) focusing on orb2 expression in a/b KCs are irrelevant to and cannot support the claim that Orb2 functions in YL neurons. *

*- Similarly, the claim (lines 302-303) that sNPF-R expression is exclusive to MB KCs conflicts with data showing effects when sNPF-R is knocked down in YL neurons. How can knocking-down a gene, which is exclusively expressed in neural population A, in neural population B affect a phenotype? This inconsistency undermines the interpretation of the results. *

*- Other examples include lines 223-227 and lines 246-249. It is very confusing how the authors came to the indications. *

- The authors also kept confusing the readers and themselves by mistakenly referring to MB KC a-lobe and YL a-lobe projection. They may know the difference between the two neural populations but they did not always refer to the right one in the text.

Response: We agree completely with the reviewer that the logic in the original manuscript was confusing and failed to clearly distinguish between the general MB Kenyon Cell (KC) population and the specific YL projection neurons. This was a major flaw, and we are grateful for the opportunity to correct it. We have undertaken a major revision of the manuscript's narrative and structure to present a clear, logical progression of discovery.

The new logical flow of the manuscript is as follows:

1. We first establish that sexual experience induces a robust, long-lasting SMD behavior that is dependent on protein synthesis
2. We then perform initial experiments to implicate the MB as a key brain region. We show that broad inhibition of MB KCs (using the ok107-GAL4 driver) disrupts SMD behavior.This result establishes the general involvement of the MB but lacks cellular specificity.
3. The remainder of the manuscript then focuses specifically on dissecting the molecular and cellular properties of these YL neurons. Finally, we have meticulously edited the entire manuscript to ensure that we always use precise terminology, clearly distinguishing between "YL neuron projections to the MB α-lobe" and the "MB KC α-lobe."

Comment 5*: The imaging figures provided are unfocused and poorly resolved, making it difficult to assess data quality. *

*- Colocalization analyses of orb2 and YL are unconvincing... Maximum intensity projection images are insufficient... complete image stacks with staining of orb2, YL, and KCs (MB-dsRed) are needed for validation. *

- Quantification of imaging data appears flawed. For example, claims of orb2 and CaMKII upregulation in MB a-lobe projections (e.g., Fig. S2F-J, Fig. 3M,N) are confounded by widespread increases in intensity across the brain, lacking specificity.

• *

*- The TRIC experiment analysis should normalize GFP signals to internal reference channel (RFP in the TRIC construct)... *

- In Fig. 6H-J, methods for counting synapse numbers are not described. How are synapse numbers counted in these low-resolution images?

Response: We sincerely apologize for the poor quality of the imaging data presented in the original manuscript. We agree with the reviewer's critiques and have taken comprehensive steps to rectify these issues.

• Image Quality: We apologize for not including the full image data in the original submission. The complete figure is now presented in revised Fig. 2J .
• Fluorescence Quantification: The fluorescence quantification has been re-analyzed. The Methods section now includes a detailed description of our protocol.
• TRIC Normalization: We apologize for not stating this explicitly in the previous version. As now described in the revised Methods subsection “Quantitative Analysis of Fluorescence Intensity”, all TRIC images were acquired with identical laser power and exposure settings. The GFP signal was background-corrected and then normalized to the RFP fluorescence encoded by the TRIC construct itself (UAS-mCD8RFP), which serves as an internal reference for construct expression and mounting thickness.

• Synapse Counting: We agree with the reviewer that the resolution of our images was insufficient for accurate synapse particle counting. We have therefore removed the problematic analysis from the former Fig 6H-J. Our conclusions regarding synaptic plasticity now rest on the more robust and quantifiable data showing a significant increase in the total area of dendritic (DenMark) and presynaptic (syt.eGFP) markers. Comment 6: The study presents data from unrelated learning paradigms (e.g., olfactory associative learning, courtship conditioning; Fig. 7) without justifying how these paradigms relate to SELTM. Particularly, the authors claimed that SELTM is related to Gr5a, which leads to appetitive memories, which involve PAM dopaminergic neurons and MB horizontal lobes. However, the olfactory associative learning with electric shock and courtship conditioning lead to aversive memories, that involve PPL1 dopaminergic neurons and the vertical lobes.

• *

Response: We thank the reviewer for requesting clarification on the rationale for including these experiments. The purpose of these assays was to test the specificity of the YL neuron circuit. A key question is whether YL neurons represent a general-purpose LTM circuit or one specialized for a particular memory modality.

The data show that knockdown of Orb2 or Nmdar2 specifically in YL neurons has no effect on the formation of LTM for aversive olfactory conditioning or aversive courtship conditioning. These negative results are critically important, as they demonstrate that the YL circuit is

not required for all forms of LTM. This finding strongly supports our revised central claim that YL neurons are specialized for processing appetitive memories derived from the specific sensory context of mating (i.e., taste and pheromonal cues from Gr5a neurons).

To improve the narrative flow of the main text, We rearranged the order of the articles. The relevant description is in lines 398-401:

“To determine whether YL neurons constitute a general LTM circuit or are dedicated to the appetitive context of mating, we tested two canonical aversive paradigms: electric-shock olfactory conditioning and courtship conditioning. If YL neurons serve as a universal LTM module, their genetic impairment should also impair aversive memory.”

lines 469-472:

“The inability of YL perturbation to impair aversive memories (Fig. 7) corroborates that this micro-circuit is dedicated to Gr5a-dependent SELTM rather than acting as a generic LTM hub”

Minor Issues

Comment 1: Fig 2F. YL projections are labeled as MBONs. Clarify whether YL neurons are the upstream or downstream (MBON) of KCs.

__Response: __Thank you for this helpful comment. As Huang et al., 2018[2] (Nat. Commun. 9:872) have mentioned, the MB093C-GAL4 driver is the MBON-α3 mushroom body output neuro. Consequently, YL neurons are positioned downstream of the MBON-α3.

We have now clarified this point in the revised manuscript lines 217-222:

“Each of these neurons extends a vertical fiber to the dorsal brain region, where they form dense arbors within the α-lobes of the mushroom body. Because the MB093C-GAL4 driver labels MBON-α3 output neuron[51], these YL arbors are positioned postsynaptically within the α-lobe and relay mushroom-body output to the anterior, middle, and posterior superior-medial protocerebrum.”

Comment 2: Extensive language polishing is required, as several sentences are unclear (e.g., lines 169-172).

Response: We apologize for the lack of clarity in the original text. The entire manuscript has undergone extensive revision and professional language editing to improve readability, precision, and grammatical accuracy.

Responses to Reviewer #2

Major Comments

Comment 1: Clearer articulation of the rationale, motivation, and significance of the overall study design and individual experiments can strengthen the manuscript and promote readership. For example, the beginnings of the abstract and introduction should define what authors mean by sexual experience-dependent long-term memory and its significance (including why it is "significant for reproductive success" (lines 46 and 92)). Similarly, employing more concrete language throughout the text will help anchor and contextualize the study. Interpretation is occasionally insufficient or does not follow directly from the data provided.

Response: We thank the reviewer for this valuable advice. We agree that the motivation and significance of our study were not articulated clearly enough. We have rewritten the Abstract and the beginning of the Introduction to address this. The revised text now explicitly defines SELTM as a protein synthesis-dependent, appetitive memory formed in response to gustatory and pheromonal cues. We explain its significance in the context of adaptive behavior, linking it to interval timing, a process by which male flies strategically adjust their mating investment (i.e., mating duration) based on prior experience to optimize reproductive success and energy expenditure. This framing provides a clearer context for our investigation into its underlying neural and molecular mechanisms.

Comment 2: Long term memory: I do not work on Drosophila memory, but a cursory search suggests that the field generally considers long term memory in Drosophila to last for 24 hr to days (courtship memory lasts for >24 hr). SMD decays between 12-24 hr after copulation. Could SMD be considered a short-term effect?

Response: This is an important point of clarification, as described in our response to Reviewer #1 (Major Comment 2), we have performed a new experiment demonstrating that the formation of SMD is blocked by the protein synthesis inhibitor cycloheximide (Figure 1I). This dependence on de novo protein synthesis is a defining characteristic of LTM, distinguishing it from short- and intermediate-term memory forms.[1] where memories lasting 12-24 hours are well-established as forms of LTM.[3] Therefore, based on both its duration and its molecular requirements, SMD represents a bona fide form of LTM.

The relevant statement is in lines 174-178:

"To determine whether the persistent reduction in mating duration (SMD) depends on de-novo protein synthesis, we fed males the translational inhibitor cycloheximide (CXM). Under this regimen, CXM completely abolished the SMD phenotype (Fig. 1I). This finding suggests that the reduction in mating investment is contingent upon the formation of LTM."

Comment 3: Fig 1B-E share the same control (naive) group. If these experiments were performed in the same replicate(s), they should be plotted in the same figure. If not, please provide more details on how experimental blocks were set up and how controls compared between replicates.

Response: Thank you for this helpful suggestion. We understand that sharing the same naive control across multiple panels (Fig. 1B–E) may raise concerns about data independence. However, we chose to present these panels separately for the following reasons:

1. Clarity and Readability: Each panel (B–E) represents a distinct temporal condition (0 h, 6 h, 12 h, 24 h post-isolation). Separating them avoids visual clutter and allows readers to focus on one time point at a time, improving interpretability.

__ Consistency with Internal Controls:__

Although the naive group is identical across panels, each experimental block (i.e., each isolation time point) was run independently on same days, with internal controls (naive vs. experienced) included in every block. This ensures that statistical comparisons remain valid within each panel, even if the naive data overlap.

We have now added a clear statement in the figure legend explaining that the naive group is shared across panels and that each time point was tested independently with internal controls. This maintains transparency while preserving the visual clarity of the current layout.

Comment 4: Serial mating (Fig 1F-H): please provide details on the methods. How much time elapsed between successive matings? Is a paired statistical test used? Sperm depletion also affects mating duration, and without this information the authors' conclusion (lines 155-156) does not automatically follow from the data.

Response:

1. __ Interval between successive matings__ We have rewritten the Methods to state explicitly that “as soon as one copulation ended the male was transferred immediately to a fresh virgin female, so the next mating began immediately.”

we add new method:

" Serial mating ____duration ____assay

Serial mating duration assay was identical to the standard procedure except that each male was presented with four DF virgin females in immediate succession: upon termination of the first copulation the male was immediately put into a fresh chamber containing the next virgin, the timer was restarted at first contact, and this step was repeated until four complete matings were recorded or 5 min elapsed without initiation, whichever came first."

__ Statistical test__

We apologize for omitting this detail. Unpaired t-test was used: for male the mating duration before (naïve) and after sexual experience was recorded, yielding paired observations. Prism’s unpaired t-test module was therefore applied to evaluate the mean difference.

The figure legend now states “with error bars representing SEM. Asterisks represent significant differences, as revealed by the Unpaired t test and ns represents non-significant difference (**p __ Mating duration versus sperm depletion__

We apologize for not having made it clear that these two observations are complementary, not contradictory. Previous work has shown that when male Drosophila copulate repeatedly, mating duration remains stable even though the number of sperm transferred—and thus the number of progeny sired—declines progressively [4]

The revised text is as follows (lines235-241):

"Previous work has shown that when male Drosophila copulate repeatedly, mating duration remains stable even though the number of sperm transferred—and thus the number of progeny sired—declines progressively. This dissociation confirms that the constant mating duration we observe in our serial-mating experiment (Fig. 1F–H) is consistent with normal sperm depletion and does not compromise the conclusion that the experience-dependent reduction in mating duration reflects long-term memory."

Thank you for helping us improve the clarity of our study.

Comment 5: Mating duration assay: Which isolation interval was chosen for the rest of the SMD experiments? The 12 hr en masse mating setup is relatively uncommon among studies on courtship/copulation/post-copulatory phenotypes, and introduces uncertainty and variability in the number and timing of matings that occurred during the 12 hr-window. This source of variability and its implication in interpreting the data should be acknowledged. Moreover, the 3 studies referenced in the methods all house males in groups of 4, whereas this study uses groups of 40. Could density confound the manifestation of SMD?

Response: We thank the reviewer for these important methodological questions.

• Isolation Interval: We have clarified in the Methods that virgin females were introduced into vials for last 1 day before assay.
• Housing Density: This is an excellent point. To control for any potential effects of housing density itself, we have clarified that our "naive" control males are also housed in groups of 40 for the same duration as the "experienced" males. Therefore, the only difference between the two groups is the presence of females, isolating the effect of sexual experience from the effect of social density. Comment 6: SMD behavior: comparing orb2 mutants and controls (Fig 1M and Fig S1K-L), loss of orb2 actually reduces the mating duration in native males (mean ~15 min) relative to controls (~20 min), and have possibly no effect on experienced males (~15 min). This is inconsistent with the SMD behavior demonstrated in Fig 1B-E. The same pattern is found for mushroom body silencing (Fig 1P, Fig S1M-N), orb2 knockdown in YL neurons (Fig 2D, Fig S2A-B), Fmr1 knockdown in YL neurons (Fig 3D, Fig S2B, S3D) and most other experiments where mating duration is not significantly different between naive and experienced males. This might demonstrate a separate role of YL neurons and its related circuit in regulating mating duration in naive males. Could the authors discuss this interpretation? As an aside, plotting genetic controls next to experimental groups is customary and facilitates comparisons between relevant groups.

Response: Thank you very much for this insightful observation.

1. Baseline differences among genotypes We agree that absolute mating duration differs slightly between genotypes (e.g. naive orb2∆/+ about 15 min vs. wild-type CS about 20 min). Such differences are common when mutations or transgenes are introduced into distinct genetic backgrounds, and they do not affect the within-genotype comparison that is the essence of SMD (sexual-experience-dependent shortening of mating duration). Therefore, for every experiment we compared naive vs. experienced males of the identical genotype, keeping all other variables constant.

Consistency of SMD across figures

In every manipulation that disrupts SMD memory (orb2∆, MB silencing, orb2-RNAi in YL neurons, Fmr1-RNAi in YL neurons, etc.) the naive–experienced difference disappears, whereas the genetic controls retain a significant ΔMD. This is fully consistent with Fig. 1B–E and demonstrates that the memory trace, not the basal duration, is abolished.

Figure layout

Following your suggestion, we have re-ordered all bar graphs so that the relevant genetic controls are placed immediately adjacent to the experimental groups, making within-panel comparisons easier.

We hope these clarifications and adjustments address your concerns.

Comment 7: Bitmap figures: unfortunately the bitmap figures are compressed and their resolution makes it difficult to evaluate the visual evidence.

Response: We apologize for the poor quality of the figures. All figures in the revised manuscript, including the scRNA-seq plots, have been remade as high-resolution vector graphics to ensure clarity and detail. For better understanding, different colored illustrations are also placed next to the scRNA-seq.

Comment 8: Sexual dimorphism of YL neurons: many neurons involved in sexual behaviors express dsx and/or fru. Do YL neurons express them?

Response: This is an excellent question. To address it, we performed a new set of experiments using genetic intersectional tools to test for the expression of doublesex (dsx) and fruitless (fru) in YL neurons. Our analysis, presented in figure supplement 2B, reveals that YL neurons are indeed fru-negative and dsx-negative. We therefore conclude that YL neurons do not belong to the canonical fru- or dsx-expressing neuronal classes and are unlikely to be intrinsically sex-specific.

We add explanation in lines 223-229:

"Our further analysis confirmed the presence of only three pairs of nuclei near the SOG in male brains, whereas female brains exhibit a greater number of nuclei near the AL (Fig. 2I), suggesting subtle sexual dimorphisms in GAL4MB093C-expressing neurons. Importantly, these neurons do not overlap with either fru- or dsx-expressing cells: co-immunostaining for GFP and Fru or Dsx revealed almost no colocalization in any brain region examined (Fig. S2B), indicating that YL neurons are distinct from the canonical sex-specific fru/dsx circuits."

Comment 9: Genetic controls for some crucial experiments are not provided, e.g. Fig 2J, Fig S3C, Fig S3E-F Fig 5B-C, F, Q-R, Fig S5A-E.

Response: We thank the reviewer for their careful attention to detail. We have now performed all the missing genetic control experiments.

Comment 10: Colocalization experiments: please provide more detail on how fluorescence is normalized for each channel across images, especially when the overall expression of an effector is up- or down-regulated after mating.

Response: We have updated the Methods section under "Quantitative Analysis of Fluorescence Intensity" and "Colocalization Analysis" to provide a detailed description of our normalization procedure.

Comment 11: Please resolve this apparent contradiction on the expression of Nmdar1 and 2 in YL neurons. On line 261: "both receptors co-expressing in Orb2-positive MB Kenyon cells"; on line 279-281 "Nmdar1 is not expressed with YL neurons [...] whereas Nmdar2 is expressed in a single pair of YL neurons..."

Response: We apologize for this contradiction, which arose from the confusing narrative structure of the original manuscript. As detailed in our response to Reviewer #1 (Major Comment 4), we have reframed the manuscript.

Comment 12: Particle analysis (Fig 6H-J): experienced males seem to have more synapses but trend towards smaller average size. It would be helpful to show number of synapses and average size as paired data, or show that the total particle area is larger in experienced males.

Response: We agree with the reviewer that this analysis was inconclusive and potentially misleading due to the limitations of image resolution. As noted in our response to Reviewer #1, we have removed this particle analysis (former Fig 6H-J) from the revised manuscript. Our claim for increased synaptic plasticity is now supported by the more robust measurement of the total fluorescence area of the pre- and postsynaptic markers, which shows a significant increase in experienced males.

Minor Comments

We thank the reviewer for their meticulous attention to detail. We have addressed all minor comments as follows:

Comment 1: 1. Some figures (e.g. Fig 3M-R) and experiments (e.g. oenocyte scRNA-seq) are not referenced in the text. dnc data is shown alongside amn and rut but the rationale for its inclusion is not provided.

__Response: __Original Fig. 3M-R (now Fig,3 M-O) was referenced on line 283. The rationale for including dnc data (as a canonical memory mutant) is now clarified in the text on lines 187-189:

"To ask whether the same molecular machinery underlies the SMD that follows sexual experience, we tested three classical memory mutants: dunce (dnc), amnesiac (amn), and rutabaga(rut)."

Comment 2: Some references might not point to the intended article (e.g. ref 123).

__Response: __The reference list has been checked and corrected.

Comment 3. Please plot genetic controls next to experimental genotypes as they are a crucial part of the experiment.

__Response: __All relevant figures now include plots of genetic controls next to experimental genotypes.

Comment 4. The "estimation statistics" plots are not necessary since the authors show individual data points. To further enhance data transparency, the authors may consider reducing the alpha and/or dot size so the individual data points are more readily visible.

Response: Thank you for this helpful suggestion! We fully agree that data transparency is essential. After carefully testing lower alpha values and smaller dot sizes, we found that either change markedly obscured the dense regions of the distributions. So we didn't change the size of the point.

The estimation-statistics overlays are kept for two courteous reasons: (i) they provide an immediate visual estimate of the mean difference and its 95 % confidence interval, which is the key statistic we base our conclusions on, and (ii) they spare readers from having to cross-reference separate tables.

Comment 5. For accessibility, please avoid using green and red in the same plot.

__Response: __We fully agree that red–green combinations can be problematic for colour-vision-impaired readers. In the present manuscript, however, the only panel that juxtaposes pure red and pure green is the Fly-SCOPE co-expression data. These scRNA-seq plots are provided only as supportive reference; the actual quantitative conclusions are based on independent genetic and imaging experiments that use magenta, cyan, yellow, and greyscale palettes. Moreover, the scope images are accompanied by detailed text descriptions of the overlapping cell clusters, so no essential information is lost even if the colours are indistinguishable

Comment 6. Fly Cell Atlas: please show color scales used for each gene as the color thresholds are gene-specific by default.The 3-color overlap on SCope also makes it very difficult to see the expression pattern for each gene. One possibility is outlining the Kenyon cells on the tSNE plots and showing the expression for each gene of interest.

Response: Thank you for this helpful suggestion. To avoid the ambiguity that arises from RGB blending in the three-colour overlay, we have added a small colour-mixing diagram next to the t-SNE plots (revised Fig. 1). This key shows the exact hues produced by pairwise and three-way overlaps:

• Red + Green = Yellow

• Red + Blue = Magenta

• Green + Blue = Cyan

• Red + Green + Blue = White

Thus, yellow, magenta or cyan dots indicate co-expression of two genes, while white dots mark cells where all three genes are detected. this diagram allows readers to interpret overlap colours at a glance without re-entering SCope.

Comment 7. Please also refer to Fly Cell Atlas as such. SCope is a visualization platform that houses multiple datasets.

__Response: __The reference to Fly Cell Atlas was added.

Comment 8. Please introduce acronyms and genetic reagents the first time they are mentioned.

__Response: __All acronyms and genetic reagents are now defined upon their first use.

Comment 9. Line 184: please specify "split-GAL4 reagents" instead of "advanced genetic tools".

__Response: __We have replaced "advanced genetic tools" with the more specific term "Split-GAL4 reagents."

Comment 10. Line 187: there are a few other lines with p>0.05 or p>0.01, so "uniquely" is inaccurate. Are the p-values in Table 1 corrected for multiple testing?

__Response: __The term "uniquely" has been revised for accuracy. No correction for multiple testing was applied because each entry in Table 1 represents a single pairwise comparison (naive vs. exp). Thus only one p-value was generated per experiment.

Comment 11. Some immunofluorescence panels lack scale bars.

__Response: __Scale bars have been added to all immunofluorescence panels.

Comment 12. Fig S2G-I: do authors mean "naive" instead of "group"?

__Response: __The term "group" in Fig S2G-I has been corrected to "naive."

Comment 13. Movie 1 should be referenced when YL neurons are first introduced.

__Response: __Movie 1 is now referenced when YL neurons are first introduced in the text.

Comment 14. Is Fig 4L similar to Fig 6L-N?

__Response: __This error has been corrected after the article was reformatted

Comment 15. Fig 7: please plot olfactory conditioning experiment results as either percentages, preference index, or paired numbers. "Number of flies/tube" is not as informative.

__Response: __Thank you for pointing this out. The bars in Fig. 7 indeed represent paired numbers, but we realise this was not stated explicitly. We apologize for the lack of clarity. In the revised manuscript we explained it in detail in figure legend and method. In the figure, we also marked the percentage of flies that chose to avoid the side of the stimulus with gas, and explained it in the Figure legend.

Reference

1. Lagasse F, Devaud J-M, Mery F. A Switch from Cycloheximide-Resistant Consolidated Memory to Cycloheximide-Sensitive Reconsolidation and Extinction in Drosophila. J Neurosci. 2009;29: 2225–2230. doi:10.1523/jneurosci.3789-08.2009
2. Huang C, Maxey JR, Sinha S, Savall J, Gong Y, Schnitzer MJ. Long-term optical brain imaging in live adult fruit flies. Nat Commun. 2018;9: 872. doi:10.1038/s41467-018-02873-1
3. Tonoki A, Davis RL. Aging Impairs Protein-Synthesis-Dependent Long-Term Memory in Drosophila. J Neurosci. 2015;35: 1173–1180. doi:10.1523/jneurosci.0978-14.2015
4. Macartney EL, Zeender V, Meena A, Nardo AND, Bonduriansky R, Lüpold S. Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster. Evol Int J Org Evol. 2021;75: 2830–2841. doi:10.1111/evo.14373

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Referee #2

Evidence, reproducibility and clarity

Summary:

Sun et al. show that Orb2-expressing, glutamatergic mushroom body neurons (YL neurons) are central to the "shorter mating duration (SMD)" behavior, where males reduce their mating duration up to 12 hours after the initial copulation. The authors use SMD as a model for understanding sexual experience-dependent long-term memory in males. A few genes implicated in long-term memory (Fmr1, CrebB) are required in YL neurons for SMD. The Nmdar-CaMKII signaling pathways is also implicated, and mating attenuates Ca2+ signaling and increases synaptic plasticity in the mushroom body and subesophageal ganglion.

Major comments:

1. Clearer articulation of the rationale, motivation, and significance of the overall study design and individual experiments can strengthen the manuscript and promote readership. For example, the beginnings of the abstract and introduction should define what authors mean by sexual experience-dependent long-term memory and its significance (including why it is "significant for reproductive success" (lines 46 and 92)). Similarly, employing more concrete language throughout the text will help anchor and contextualize the study. Interpretation is occasionally insufficient or does not follow directly from the data provided.
2. Long term memory: I do not work on Drosophila memory, but a cursory search suggests that the field generally considers long term memory in Drosophila to last for 24 hr to days (courtship memory lasts for >24 hr). SMD decays between 12-24 hr after copulation. Could SMD be considered a short-term effect?
3. Fig 1B-E share the same control (naive) group. If these experiments were performed in the same replicate(s), they should be plotted in the same figure. If not, please provide more details on how experimental blocks were set up and how controls compared between replicates.
4. Serial mating (Fig 1F-H): please provide details on the methods. How much time elapsed between successive matings? Is a paired statistical test used? Sperm depletion also affects mating duration, and without this information the authors' conclusion (lines 155-156) does not automatically follow from the data.
5. Mating duration assay: Which isolation interval was chosen for the rest of the SMD experiments? The 12 hr en masse mating setup is relatively uncommon among studies on courtship/copulation/post-copulatory phenotypes, and introduces uncertainty and variability in the number and timing of matings that occurred during the 12 hr-window. This source of variability and its implication in interpreting the data should be acknowledged. Moreover, the 3 studies referenced in the methods all house males in groups of 4, whereas this study uses groups of 40. Could density confound the manifestation of SMD?
6. SMD behavior: comparing orb2 mutants and controls (Fig 1M and Fig S1K-L), loss of orb2 actually reduces the mating duration in native males (mean ~15 min) relative to controls (~20 min), and have possibly no effect on experienced males (~15 min). This is inconsistent with the SMD behavior demonstrated in Fig 1B-E. The same pattern is found for mushroom body silencing (Fig 1P, Fig S1M-N), orb2 knockdown in YL neurons (Fig 2D, Fig S2A-B), Fmr1 knockdown in YL neurons (Fig 3D, Fig S2B, S3D) and most other experiments where mating duration is not significantly different between naive and experienced males. This might demonstrate a separate role of YL neurons and its related circuit in regulating mating duration in naive males. Could the authors discuss this interpretation? As an aside, plotting genetic controls next to experimental groups is customary and facilitates comparisons between relevant groups.
7. Bitmap figures: unfortunately the bitmap figures are compressed and their resolution makes it difficult to evaluate the visual evidence.
8. Sexual dimorphism of YL neurons: many neurons involved in sexual behaviors express dsx and/or fru. Do YL neurons express them? If they do, they might be a subset of characterized and named dsx/fru neurons.
9. Genetic controls for some crucial experiments are not provided, e.g. Fig 2J, Fig S3C, Fig S3E-F Fig 5B-C, F, Q-R, Fig S5A-E.
10. Colocalization experiments: please provide more detail on how fluorescence is normalized for each channel across images, especially when the overall expression of an effector is up- or down-regulated after mating.
11. Please resolve this apparent contradiction on the expression of Nmdar1 and 2 in YL neurons. On line 261: "both receptors co-expressing in Orb2-positive MB Kenyon cells"; on line 279-281 "Nmdar1 is not expressed with YL neurons [...] whereas Nmdar2 is expressed in a single pair of YL neurons in both male and female brains".
12. Particle analysis (Fig 6H-J): experienced males seem to have more synapses but trend towards smaller average size. It would be helpful to show number of synapses and average size as paired data, or show that the total particle area is larger in experienced males.

Minor comments:

1. Some figures (e.g. Fig 3M-R) and experiments (e.g. oenocyte scRNA-seq) are not referenced in the text. dnc data is shown alongside amn and rut but the rationale for its inclusion is not provided.
2. Some references might not point to the intended article (e.g. ref 123).
3. Please plot genetic controls next to experimental genotypes as they are a crucial part of the experiment.
4. The "estimation statistics" plots are not necessary since the authors show individual data points. To further enhance data transparency, the authors may consider reducing the alpha and/or dot size so the individual data points are more readily visible.
5. For accessibility, please avoid using green and red in the same plot.
6. Fly Cell Atlas: please show color scales used for each gene as the color thresholds are gene-specific by default.The 3-color overlap on SCope also makes it very difficult to see the expression pattern for each gene. One possibility is outlining the Kenyon cells on the tSNE plots and showing the expression for each gene of interest.
7. Please also refer to Fly Cell Atlas as such. SCope is a visualization platform that houses multiple datasets.
8. Please introduce acronyms and genetic reagents the first time they are mentioned.
9. Line 184: please specify "split-GAL4 reagents" instead of "advanced genetic tools".
10. Line 187: there are a few other lines with p>0.05 or p>0.01, so "uniquely" is inaccurate. Are the p-values in Table 1 corrected for multiple testing?
11. Some immunofluorescence panels lack scale bars.
12. Fig S2G-I: do authors mean "naive" instead of "group"?
13. Movie 1 should be referenced when YL neurons are first introduced.
14. Is Fig 4L similar to Fig 6L-N?
15. Fig 7: please plot olfactory conditioning experiment results as either percentages, preference index, or paired numbers. "Number of flies/tube" is not as informative.

Significance

The manuscript describes an extensive and comprehensive set of experiments aimed at elucidating the role of a subset of mushroom body neurons in mediating a male post-mating sexual behavior, which the authors use as a model for sexual experience-dependent long-term memory. Long-term post-mating responses in females have been well characterized in Drosophila and other insects, but post-mating long term memory in males are less well understood despite a few studies reporting their importance in mating success. How males adjust their mating duration based on internal and external cues can reveal insights about decision making and interval timer mechanisms. This study represents a functional advancement in the neuronal and molecular mechanisms of how memory and experience regulates a sexual behavior in male Drosophila. Overall, the manuscript can significantly benefit from general editing on clearer articulation of rationale and more appropriate interpretations of data. Higher resolution versions of bitmap figures is also crucial. The SMD experiments invite an alternative interpretation of data that centers on YL neurons' role on regulating mating duration in naive males, which alongside other roles of the mushroom body demonstrated in this manuscript, could add more depth to the study.

The findings in this manuscript will be of interest to a specialized audience interested in memory, neural circuits of behavior, and Drosophila sexual behavior. I work on Drosophila sexual behavior and circuits, but lacking experience on memory research, I am not as familiar with the mushroom body and conditioning experiments.

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

This study explores the molecular and neural circuitry mechanisms underlying sexual experience-dependent long-term memory (SELTM) in male Drosophila. The authors use behavioral, imaging, and bioinformatics approaches to identify YL neurons, a subset of mushroom body (MB) projecting neurons, as crucial for SELTM formation. They propose that YL neurons receive inputs from WG neurons via the sNPF-sNPFR pathway and implicate molecular players such as orb2, fmr1, MDAR2-CaMK, and synaptic plasticity in their function.

However, the evidence presented does not adequately support the authors' claims. The data fail to cohesively tell a logical story, and key conclusions appear to be based on assumptions and correlations rather than robust evidence.

Major comments:

1. The study identifies the knowledge gap (lines 103-104) but fails to integrate relevant literature, particularly Shohat-Ophir et al., Science (2012), and Zer-Krispil et al., Curr Biol (2018). These studies established that ejaculation induces appetitive memory in male Drosophila via corazonin and NPF neurons. The current study does not provide direct evidence that the "act of mating itself" drives SELTM, as it includes both courtship and copulation.
2. The nature of the observed long-lasting reduced mating duration requires clearer characterization: Is this an associative memory or experience-dependent behavioral plasticity? Can the formation of this long-term memory be blocked by protein synthesis inhibitors, such as cycloheximide?
3. While schematics illustrate the working hypotheses, the text lacks detailed explanations, leaving the reader unclear about the rationale behind certain conclusions.
4. The logic to draw certain conclusions was confusing and misleading.
   • For instance, the role of orb2 in SELTM is examined via knockdown in MB Kenyon cells (KCs) (using ok107>orb2-RNAi), which is irrelevant to the claim that orb2 functions in YL neurons. Additionally, RNAseq analyses (Fig. 1N-S) focusing on orb2 expression in a/b KCs are irrelevant to and cannot support the claim that Orb2 functions in YL neurons.
   • Similarly, the claim (lines 302-303) that sNPF-R expression is exclusive to MB KCs conflicts with data showing effects when sNPF-R is knocked down in YL neurons. How can knocking-down a gene, which is exclusively expressed in neural population A, in neural population B affect a phenotype? This inconsistency undermines the interpretation of the results.
   • Other examples include lines 223-227 and lines 246-249. It is very confusing how the authors came to the indications.
   • The authors also kept confusing the readers and themselves by mistakenly referring to MB KC a-lobe and YL a-lobe projection. They may know the difference between the two neural populations but they did not always refer to the right one in the text.
5. The imaging figures provided are unfocused and poorly resolved, making it difficult to assess data quality.
   • Colocalization analyses of orb2 and YL are unconvincing, especially given that orb2 is well-documented in literature as expressed in MB a-KCs and YL projection wrapping MB a-lobe. Maximum intensity projection images are insufficient for confirming colocalization; complete image stacks with staining of orb2, YL, and KCs (MB-dsRed) are needed for validation.
   • Quantification of imaging data appears flawed. For example, claims of orb2 and CaMKII upregulation in MB a-lobe projections (e.g., Fig. S2F-J, Fig. 3M,N) are confounded by widespread increases in intensity across the brain, lacking specificity.
   • The TRIC experiment analysis should normalize GFP signals to internal reference channel (RFP in the TRIC construct), as per established protocols in the original paper.
   • In Fig. 6H-J, methods for counting synapse numbers are not described. How are synapse numbers counted in these low-resolution images?
6. The study presents data from unrelated learning paradigms (e.g., olfactory associative learning, courtship conditioning; Fig. 7) without justifying how these paradigms relate to SELTM. Particularly, the authors claimed that SELTM is related to Gr5a, which leads to appetitive memories, which involve PAM dopaminergic neurons and MB horizontal lobes. However, the olfactory associative learning with electric shock and courtship conditioning lead to aversive memories, that involve PPL1 dopaminergic neurons and the vertical lobes.
7. Some figures are not referred to in the text. For example, Fig S1 K and L (also, what's the difference between these two figures?) and Fig 3M-R. What is MB-V3 in Fig 4J-K?

Minor issues

1. Fig 2F. YL projections are labeled as MBONs. Clarify whether YL neurons are the upstream or downstream (MBON) of KCs.
2. Extensive language polishing is required, as several sentences are unclear (e.g., lines 169-172).

Significance

This study potentially advances our understanding of how sexual experience modifies future mating behaviors. While previous work has shown that mating induces appetitive memory in males, the mechanisms linking this memory to future mating behavior remain poorly understood. This work could provide valuable insights into these mechanisms, pending appropriate revisions.

His supervisor thought their was nothing wrong

This passage emphasizes the way film responded to the wider cultural modernization of the early 1900s through technology, realism, and emotional involvement. The employment of camera movement and editing registered new conceptions of seeing and experiencing space and time, mirroring the break-throughs in science and psychology. Film therefore became a signifier of modernity, transforming visual entertainment into an influential art form expressive of human complexity and social transformation.

This part shows that the crisis wasn’t just social but emotional. Uncertainty caused peoples strife after the war. Even the universe seemed unstable. This new worldview influenced all types of art, suggesting that truth is known and knowledge is limited.

This reminds me about a recent online controversy in my community where a very famous hijab brand's owner had a previous racist picture resurface online. Even though she had apologized for it multiple times the people of the black muslim community don't feel comfortable buying from her because of her previous actions. Which brings me to my point that digital footprint doesn't ever leave no matter how private it may seem.

Can we turn this featured section off for launch and have the events calendar go straight across?

Can these each link to the event pages?

Social media companies have a lot to gain from selling private data. They can sell it to as many as are willing to pay, money being the main incentive. Companies and corporations are willing to spend a lot of money to get to know data about their potential customer, if they know the majority of their potential customers are x, then they will respond by catering to x. Do a lot of people face similar problems? Luckily for them WebSiteName has just the solution!

I have a lot of fears around privacy on social media. I worry that social media companies are constantly listening and tracking what I say and do. I worry that they have a profile on me with everything about me. And I believe they are doing these things because I will be talking about something with my friends or parents, and then I will get an ad for it on instagram. I didn't plug anything into instagram to make them know that I was thinking about it yet they will somehow know. This is the kind of thing I don't like about social media.

At the moment there are still a good amount of legal protections that restrict companies from being too flagrant with your privacy but over the lat few decades that has become more and more of a market is companies actually selling people's information. I also think that people don't like the idea of a company having all their information so maybe another thing stopping them is the public sentiment toward private companies doing these massive privacy violations. But there is a lot of money on the other side of that fence and more and more and more companies are deciding that they want to take that leap and violate privacy.

As faculty who are considering what it means to bring OEP into our classrooms in this particular moment (fall of 2025) this note about perpetrating harm unintentionally resonates with our conversations. Specifically, how do we balance the risks that are changing and evolving for putting information into the public sphere (eg. depending on your context individuals can be targeted)? What does the evolution of how AI uses our information that's publicly available mean for the risks of doing OEPs in our classes?

How the Trump administration is dramatically reshaping education in America<br /> by [[John Yang]] of PBS at PBS News Weekend accessed on 2025-10-22T12:01:15

Interview with Jennifer Smith Richards of ProPublica

Anti-DEI, Anti-CRT bills

The magical source behind your favorite pastrami sandwiches and chili dogs<br /> by [[By Josh Heller]] for LAist<br /> accessed on 2025-10-22T11:56:41

This article talks about the right to privacy, and everyone in this country has the right to privacy on the internet. The internet should protect users' privacy. This right encourage more users to participate in any social media. I personally agree with this right, and I feel safe with the right to privacy.

This connects to not having a fixed mindset when doing research. During the research process it is important to test your original hypothesis because you never know what alternative solution you could come up with after the previous prediction.

It is especially important for science to be shared freely. This is because it will help people form networks with each other to solve complicated solutions during these difficult times of our society.

It seems like there is an overlap where some people think social media is news media, and they believe everything they read on social media as the real news sometimes.

its in truth a deep analysis and the the way or articulation that is to be considred as a critical thinking stage

there has been a change over time in how the media has passed the informantion and the changes explain the differences between the articles

I think it’s interesting how the video points out that ADHD is found from kids before they are 12. I would think if symptoms start coming in later closer to puberty, it wouldn’t be different. I would even think it’s possible for younger kids to have mask traits such as procrastination due to the help of their parents so it might spark more obvious signs when they reach adulthood when there are less people helping them succeed.

This made me curious, are there any programs trying to fund science just for science's sake, or are all the funders profit based?

I have never thought about science as being inherently social. I think this idea is contradictory to the personality type and abilities most people associate with scientists.

https://www.instagram.com/nprfreshair/reel/DNVlf2tMstg/?hl=en

Terry Gross reading a book has rendered it useless for others to read.

Dog earing of top corner for interesting sections or questions she may have for the author.

Dog earing bottom corner as an indicator of remembering facts for the intro or for sentences she wants to quote.

Uses front of book for connecting themes and focus, so she won't forget it.

Introductions/prologues for quick overviews of what the book is about and why they wrote it.

I think it’s strange that disabled people aren’t able to design for themselves considering they have first hand experience with most of the issues. One of the first parts of designing is to empathize with a certain demographic so most disabled people would be able to understand the problems. I don’t get how you would need higher qualifications for someone designing a ramp as a disabled person rather than a non-disabled designer.

Colonial soldiers often joined for short times. The army made them stay longer, which made some unhappy with British control.

Clough is talking about the French and Indian War, when the British and colonists fought the French and Native Americans for land in North America.

Ye” just means “the.” People used it in old English writing.

Sounds like pedagogy. I think i'm still not getting the difference between inclusive design and universal design for learning.

studying the family life cycle will help with creating family interventions (Purpose of article???)

Still not much research on the impact of extended family on adolescent mood :(

Purpose for the article (???)

This info can improve the help black people get in therapy

Broadening our research and definition of families could reduce some of the negative effects extended family may have regarding socioeconomic status

This is going in the conclusion, all 4 sources back this up.

the negative effects aren't tied to race, but socioeconomic status (which can be tied back to race in some ways but getting into that gets us off track of the research question)

This is the only negative effect I've seen for children/adolescents

The negative effects seem to be geared more towards the adults than the adolescents

Black and White families can't be compared the same way

Affirm the importance of extended family in single parent homes

The benefits of extended family are shown clearly through black families based off origin, values, beliefs, and varying familial structures that are distinct from the white "standard"

researchers turned their focus to tradition and culture, and less of the white "norm"

The main problems of research on black families based on the nuclear definition was caused by assumptions that a) all black people will possess cultural attributes to the same degree, b) research on lower socioeconomic black families represented the entire black population (they generalized all of their research) and c) That white and black people had a cultural equivalence and that their behaviors could be compared the same way based off of a common standard

not related to my question, just guessing that this resulted in harmful stereotypes for black people (now that I think about it maybe it is related because the results would've been different if they didn't skew the data)

skewed results because they forced black families into their standards

These familial bonds have importance, but because they aren't included in the "standard" definition, research on the effects are limited

is he Lenoro-Otero suggesting that there's a connection between diverse family structure and diverse cultures and ethnicities?

This will cause many families to deviate from the "norm", the "standard" definition will exclude many families in the US that may not be white or middle to upperclass

aligns with how other sources explained the changes in our definition of family overtime

Black Americans have different family life (close with extended family) than White Americans. Their norms are seen as atypical and divergent from the "norm", causing limited research on the effects of extended family on Black Americans

1. Increased access2. Reduced friction3. More emotional contextThe impact of inclusive design is morethan just the products that people use. It’salso a shift in our mindset, methods, andbehaviors.

Realm of psychology, spirituality, libera; arts/

New Yorker Staff. 2007. “The Typing Life: How Writers Used to Write.” The New Yorker. https://www.newyorker.com/magazine/2007/04/09/the-typing-life (October 22, 2025).

typewriter plays a central role in the story

source? original quote?

via Darren Herschler-Henry's book

Good example of basic Teams bot interaction

Interesting option for showcasing capabilities / FAQ, etc. without needing to manage a separate website.

https://www.reddit.com/r/typewriters/comments/1o8o98j/comment/njx2pgo/?context=1

Haul Out the Halloween (2025), Hallmark Movie

This not only affects their health but also their education. If a student cannot afford to buy glasses they will likely continue to struggle seeing the board in school. Similarly if a student has a hard time hearing, they will face disadvantages which can affect their academic performance. It is unfair for intelligent students to fall behind because they can't afford the luxury of getting medical attention.

Many, like my family have come in the pursuit of the American Dream. While I do think it's possible, it is much easier said than done, especially for those who come from a low-income. Not everyone has access to resources that can help guide those new to this country. Because of that, many left to fend for themselves, not knowing what is necessary to set themselves for success. Yes, many can find employment, but employment does not equate to the American dream.

UPDATED Watch Woody Allen's Interview with Bill Maher: "Sunset Boulevard" (the Movie) is "Fun Junk," "Streetcar" is "Perfect" - Showbiz411<br /> by [[Roger Friedman]]<br /> accessed on 2025-10-22T10:20:51

AKA, LLM prompt: make it sound academic.

It's interesting how people so early on in our lives play a huge role in our future achievement. I see this being true in many aspects. Hearing my teachers talk about their higher education experience inspired me to do the same. Not only do educators play a huge role in influencing students for higher education, but often times help create a welcoming and safe environment for students who don't feel safe anywhere else. Because of this I strongly believe elementary school and its educators are what shape student success.

I have always wondered what the implications are from parents who work all the time and have little time to see their kids. I have seen this on both sides, low-income students whose parents work a lot to survive, and well-off students whose parents are always on business trips they rarely see their kids. While both are done for different reasons, I think the experience of the kids is similar emotionally.The lack of a parent figure in the home leads many to become independent. While the experiences can be different, I find many of the students in similar situations are able to find a common ground In how they felt, but plan to go about their lives in different ways. Low-inccome students often want a well paying job that allows them to see their kids, while wealthier students follow a similar path made by their parents.m

facts are boring but overall good

good shows a compare and contrast

good something that is relatable

shows what to use and how to use it

This is the introduction to the paper, and discusses the main problem that the research aims to solve.

This sentence clearly states the author's main argument: single stories create stereotypes, and they can be challenged through real interaction.

This section introduces the source (Adichie’s TED Talk), explains her experience, and sets the stage for the main idea by showing how a “single story” can limit perception.

This engages the reader by asking a reflective question and raising curiosity about assumptions and stereotypes.

thesis

background

Although I was always fond of art projects in preparation for mother and father's day, I always felt bad for the students who did not have one or the other. While it is important to teach young students the importance of appreciation and giving gifts during an important day, it can make others feel uncomfortable and secluded from the rest. During my time working on such crafts, I never realized there could be students next to me who were participating just to fit in, but had no one to give the gift to.

I dont think it is necessarily their fault they are unaware of others situation. There is a lack of awareness and lack of emphasis on educating others about financial hardships many face. Many are oblivious to the fact that someone could be living in poverty because they have never encountered someone in that situation. It is unfair to judge those who are unaware, rather it's important to focus on the education system which is meant to inform students but often fails to do its job.

This is unfortunately true for many, and became clear to me when I came to university. Many first-generation students and students who come from poverty have been too worried figuring out how to pay for college or simply how to survive, which forces them to take time away from hanging out with friends and exploring their own curiosity. This is why many of these students either go crazy their first year or feel as if they don't belong.

This is a reality for many people, especially across California, on of the most expensive states to live in. However, I don't believe this accounts for changes in income. Yes, if someone living in California were to move to a state like Kentucky they'd likely have the financial stability to live well. But, as time continues and people settle in, they'll be forced to work the wages within that state, not California. This can potentially cause financial hardships because of the sudden change in lifestyle. Although it could be beneficial to move out of state, it is important to be cautions.

Q3. Kaitlin Breuchel Ball and Loewe’s ideas connect well with “Only Geniuses Can Be Writers.” Both challenge the belief that writing is something only a few talented people can do. They remind us that everyone can be a writer and that creativity shows up in many different kinds of writing, not just in what’s seen as “artistic.”

Q4. Kaitlin Breuchel In concluding their argument, Ball and Loewe assert that there is creativity in all writing, and not just what we term "creative writing." They argue that setting a definition for creativity to be used in some forms of writing makes people imagine and thought that enter ordinary writing. From what I have read, I view "creative writing" differently i think it cannot just be referring to poetry or fiction but any form of writing where a person is deciding, communicating ideas, and speaking with others.

Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Ecology.<br /> See the peer reviews and the recommendation.

the first priniciple that the author's argue that contribute to a successful multinational federation is Staatsvolk. it's not a pancea but the evidence demonstrates that the more heterogenous a federation is the more likely it will be unstable, face secessionism, or break up becuase the minorities are more likely to think they can prevail. the authors suggest that multinational federations without staatsvolk to survive as democratic entites they must develop consociational practices to protect interests of all comm. adding Gannon into this paragraph because they share similar views, the majority nation must appropriately behave to maintain stability.

mentions india's refusal to recognize religion, not ethnicity, as the basis of state formation. india is a successful multinational federation but due to their refusal to recognize religion they have had issues with Kashmir and Punjab. violoence would be a result of centralising decisions.

canada is a great example of a successful multinational federation that has had its up and downs regarding quebec. the problems arose as the authors argue, due to centralising movements away from multinational federalist principles. in 1982 the canadian prime minister Trudeau introduced two major changes to the constituion to weaken Quebec's powers. tensions rose and a there was a close referendum result in 1995 that lessened the tensions.

introduces the idea that these failed multinational federations had a lack of Staatsvolk and were the most heterogenous. this combine created major tensions. in O'Leary's past research they have found that the world's stable and durably dem majoritatian federatiosn are so because that have straatsvolk (dominant people/identity)

authors argue that collapses were caused by insufficient implementation of pluri-national federal principles. for example war started in croatia because the serb population wanted to stay in Yugoslavia (this sentiment also spread to bosnia).

the authors even argue that it is because of these centralising movements and majoritarian policies of the dominant groups that contribute to the wars and conflict of the existing nations that came out of the federations.

wrong historical causation leads to prominent arguments against multinational federation. the authors in this article argues its attempts to unitarise and centralise multinational federations that lead to secession and violence. For Yugoslavia, the successive Serbian-dominated moves against autonomy of the others lead to Kosovo to de facto breakaway. it could be said that federal constitutions with procedural and negotiable secession rules might have avoided violence and even succession better.

the authors of this paper counters the argument that mono-nation-building strategies can be used as an alternative for deeply diverse states is that these strategies have not been successful. UK's civic and unitary state did not prevent the nationalism of its different nations. Therefore the UK had to use a devolution strategy but it still does not quell nationalism.

counterfactuals = relating to or expressing what has not happened or is not the case. authors against multinational federation argue that it was unnecessary to accommodate diversity through federation and that there were democratic civic or unitarists (one nation) alternatives.

the econoic systems within these failed federations eventually proved incapable of providing a reasonable or growing standard of living for citizens. different economic systems of the different regions of the state caused resentment.

colonial federations still were imprinted by the departing metropolitan. s decision to federate instead of the indigenous elites. this case includes Nigeria and Cameroon.

Yugoslavia isn't actually a multinational federation, it was decentralised but that doesn't mean its was democratic, it was held together by the League of Communists. Other "multinational federations" which are more like pseudo-federations include USSR, Czechoslovakia, and Nigeria. they all had weak or no overarching identities and no democratic mechanism for developing those identities

this harks back to an article i read a little bit of (i think it was the Yugoslavia one) where america's motivation relied on ideological differences to further break up that federation. but that's an interpretation. regarding the paragraph, american academic argue that the break-up of former communist federations are due to their implemenation of "ethno-federal" strcutures. Jack Snyder argues that ethnofederalism tends to heighten and politicise ethnic consciousness, creating self-conscious intelligentsia and org strucutres of an ethnic state in waiting. implying that federalism leaves ethnic groups waiting for something they will not receiving leading to nationalism and tensions. additionally snyder notes that nationalist violence happened only where ethnofederal institutions channelled pol activity along ethnic lines (ex: USSR and Yugoslavia).

brings in the disagreement that american academy and policy making have with multinational federations that is unlike Jacobins because americans reject the strong state idea. The disagreement is that America's position on federalism is that it shoud be more national than pluri-national. Instead american praise federalism for its ability to diffuse power to multiple points and it protects liberalism and enhances markets. americans even encouraged federalism for post-war germany. additionally, american federalism's goal is the same as Jacobinism which is to contruct a single poeple out of many.

breaksdown where the negativity toward multinational federations are coming from. Specifically from France's Jacobins where he thinks that pol recognition of multiple nations/ethnic comm institutionalizes and reinforces division, endangers national/state unity, and leads to state breka up. Then refers to practical examples which include several eastern euro states that have moved to replacing pluri-naitonal federations with "nationalising" states (tightly centralised, controlled by dom national comm, and intent on honogenisation of deviant identities). these nationalist seek indep as unitary, sovereign and indivisible nation-staets with some able to consider confederation.

highlights the federal principle of the ability for groups to easily secede if they want to. in the case of Yugoslavia is was a part of the full constitutent unit of the USSR were able to break away and break even further into independent states. In the case of succession it begins when the regional gov balme their central counterpart for whatever ails them; this contributes to intergovernmental politics of division

details the accounts were multi-national federations have collapsed or have failed to be durably democratic all around the world.

understanding the term and relevance of "multination/pluri-national" or "ethno-federal" federations instability. mentions Yugoslavia's disintegration during its transition to democracy. (which the authors argues is crucial for the stability of multinational federations, alongside the choices of no democracy and no single state)

argues that multinational federations cna succeed under certain conditions and the arguments against are greatly exagerated, based on majoritarian bias, spurious arguments, and misleading comparisons.

The two laws. And isn't there one in Washington?

social history of the above

Geography, infrastructure, history policy cf Bangkok floodplain - People shoudl be left with an overview of the situation Bkk faces vis-a-vis climate flooding hydrology "broader stakes" of innovation

Stronger alternatives: The central claim that, "AI-powered systems enable teams collaborate, explore new choices, and develop ideas in real time," is a good argument, however I would end the sentence with a stronger explanation. The ending of their sentence is vague and indistinct.

Consistency: This article is consistent, drawing all conclusion back to the thesis that AI improves firms. It also recognizes that AI needs to be implemented with care. By doing this the article is recognizing the counter claim that AI is "taking over," which improves the logic of the argument.

Ambiguity: The term "operations efficiency" is used quite a bit in this article. An explanation of what operations efficiency really means should be provided so that the reader understands the full context of the argument. Operations efficiency could refer to refer to product development, marketing, or sales.

Fallacies: I couldn't find a fallacy in this article. Evidence is used to support the claim, there aren't emotional/personal attacks, and evidence is broken down and explained. This article is thoroughly written with the intent to inform, not attack.

Validity: This article uses both data and reasoning to support the claim. It gives numerous examples and statistics of how AI betters firms. Each piece of evidence is brought back to the claim that AI is maximizing innovation and creativity.

Soundness: The evidence this article uses is true and relavant. I know it's true because they provide data and statistics from an analysis that support the claim. This evidence connects to the claim that AI increases innovation, which is a prime aspect of the thesis.

Link your ethics to your plans to grow the business sustainably--this allows you to both articulate your values and end with a hopeful note about the future. It's a good move that helps both describe and promote your vision.

Second-order headings (reflected in the Table of Contents) and bulleted lists help break up the clutter of the document.

You may wish to consider some bulleted lists to make the examples stand out more visually.

For each value, list at least one practice that demonstrates that value. It need not be long, but it does make your commitment clearer to the reader of the document.

The story about COVID can demonstrate qualities like resilience and determination, which you can then describe as qualities that drive your business model.

So, tldr, you are always linking your company's actions and your company's values.

Can you briefly tell us what was wrong with the existing ones that created this opportunity for you to make a better (and more ethical) product? What did the opportunity look like?

Use the "insert table of contents" function to create a symmetrical presentation with consistent columns.

eLife Assessment

This fundamental study presents a new method for longitudinally tracking cells in two-photon imaging data that addresses the specific challenges of imaging neurons in the developing cortex. It provides compelling evidence demonstrating reliable longitudinal identification of neurons across the second postnatal week in mice. The study should be of interest to development neuroscientists engaged in population-level recordings using two-photon imaging.

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

None major. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision

Comments on revisions:

I have no further requests. I think this is an excellent manuscript

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths

The authors have satisfactorily addressed the majority of our questions and comments, and the revisions substantially improve the manuscript. The expansion of Track2p to accept general NumPy array inputs makes the tool more accessible to researchers using different analysis pipelines. While the absence of benchmarking standards remains a limitation across the field, the release of the ground-truth dataset is an important step forward that will allow other researchers to evaluate and compare algorithms.

Minor point

(1) The authors tested the robustness of the algorithm across non-consecutive days. As expected, performance drops significantly under these conditions. We agree that this limitation reflects biological constraints due to brain growth rather than shortcomings of the algorithm itself. This is relevant for researchers planning to use Track2p for longitudinal imaging or benchmarking new algorithms, and we recommend including some of this information in the Supplementary Information along with a brief discussion.

Comments on revisions:

We acknowledge the extended documentation for using Track2p and converting between Suite2p outputs and NumPy arrays. This addition is of great utility. We would also suggest further expanding the documentation for the NumPy array implementation, as we ran into some errors when testing this feature using NumPy arrays generated from deltaF traces, TIFF FOVs, and Cellpose masks.

Reviewer #3 (Public review):

Summary:

In this manuscript Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks, however, tools to study them longitudinally at a single cell level are scarce. This paper provides a valuable approach to study both single cell dynamics across days and state-drive network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show feasibility to track populations of neurons in the same mice capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

Major points

The authors use a viral approach to label cortical interneurons. It is unclear how Track2P will perform in dense networks of excitatory cells using GCaMP transgenic mice.

The authors used 20 neurons to generate a ground truth data set. The rational for this sample size is unclear. Figure 1 indicates capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

It is unclear how movement was scored in the analysis shown in Fig 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement or was movement quantified as amount of time in which the treadmill was displaced?

The rational for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

Comments on revisions:

The authors have addressed carefully all my comments. This is an interesting paper.

Author response:

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

Reviewer #1 (Public review):

We thank the reviewer for very enthusiastic and supportive comments on our manuscript. 

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

No major weaknesses were identified by this reviewer. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision.

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled the identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths:

The manuscript is well written, and the analysis pipeline is clearly described. Moreover, the dataset used for validation is of high quality, considering the technical challenges associated with longitudinal two-photon recordings in mouse pups. The authors provide a convincing comparison of both manual annotation and "CellReg" to demonstrate the tracking performance of "Track2p". Applying this tracking algorithm, Majnik and colleagues characterized hallmark developmental changes in spontaneous network activity, highlighting the impact of longitudinal imaging approaches in developmental neuroscience. Additionally, the code is available on GitHub, along with helpful documentation, which will facilitate accessibility and usability by other researchers.

Weaknesses:

(1) The main critique of the "Track2p" package is that, in its current implementation, it is dependent on the outputs of "Suite2p". This limits adoption by researchers who use alternative pipelines or custom code. One potential solution would be to generalize the accepted inputs beyond the fixed format of "Suite2p", for instance, by accepting NumPy arrays (e.g., ROIs, deltaF/F traces, images, etc.) from files generated by other software. Otherwise, the tool may remain more of a useful add-on to "Suite2p" (see https://github.com/MouseLand/suite2p/issues/933) rather than a fully standalone tool.

We thank the reviewer for this excellent suggestion. 

We have now implemented this feature, where Track2p is now compatible with ‘raw’ NumPy arrays for the three types of inputs. For more information, please check the updated documentation: https://track2p.github.io/run_inputs_and_parameters.html#raw-npy-arrays. We have also tested this feature using a custom segmentation and trace extraction pipeline using Cellpose for segmentation.

(2) Further benchmarking would strengthen the validation of "Track2p", particularly against "CaIMaN" (Giovannucci et al., eLife, 2019), which is widely used in the field and implements a distinct registration approach.

This reviewer suggested  further benchmarking of Track2P.  Ideally, we would want to benchmark Track2p against the current state-of-the-art method. However, the field currently lacks consensus on which algorithm performs best, with multiple methods available including CaIMaN, SCOUT (Johnston et al. 2022), ROICaT (Nguyen et al. 2023), ROIMatchPub (recommended by Suite2p documentation and recently used by Hasegawa et al. 2024), and custom pipelines such as those described by Sun et al. 2025. The absence of systematic benchmarking studies—particularly for custom tracking pipelines—makes it impossible to identify the current state-of-the-art for comparison with Track2p. While comparing Track2p against all available methods would provide comprehensive evaluation, such an analysis falls beyond the scope of this paper.

We selected CellReg for our primary comparison because it has been validated under similar experimental conditions—specifically, 2-photon calcium imaging in developing hippocampus between P17-P25 (Wang et al. 2024)—making it the most relevant benchmark for our developmental neocortex dataset.

That said, to support further benchmarking in mouse neocortex (P8-P14), we will publicly release our ground truth tracking dataset.

(3) The authors might also consider evaluating performance using non-consecutive recordings (e.g., alternate days or only three time points across the week) to demonstrate utility in other experimental designs.

Thank you for your suggestion. We have performed a similar analysis prior to submission, but we decided against including it in the final manuscript, to keep the evaluation brief and to not confuse the reader with too many different evaluation methods. We have included the results inAuthor response images 1 and 2 below.

To evaluate performance in experimental designs with larger time spans between recordings (>1 day) we performed additional evaluation of tracking from P8 to each of the consecutive days while omitting the intermediate days (e. g. P8 to P9, P8 to P10 … P8 to P14). The performance for the three mice from the manuscript is shown below:

Author response image 1.

As expected with increasing time difference between the two recordings the performance drops significantly (dropping to effectively zero for 2 out of 3 mice). This could also explain why CellReg struggles to track cells across all days, since it takes P8 as a reference and attempts to register all consecutive days to that time point before matching, instead of performing registration and matching in consecutive pairs of recordings (P8-P9, P9-P10 … P13-P14) as we do.

Finally for one of the three mice we also performed an additional test where we asked how adding an additional recording day might rescue the P8-P14 tracking performance. This corresponds to the comment from the reviewer, answering the question if we can only perform three days of recording which additional day would give the best tracking performance. 

Author response image 2.

As can be seen from the plot, adding the P10 or P11 recording shows the most significant improvement to the tracking performance, however the performance is still significantly lower than when including all days (see Fig. 4). This test suggests that including a day that is slightly skewed to earlier ages might improve the performance more than simply choosing the middle day between the two extremes. This would also be consistent with the qualitative observation that the FOV seems to show more drastic day-to-day changes at earlier ages in our recording conditions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks; however, tools to study them longitudinally at a single-cell level are scarce. This paper provides a valuable approach to study both single-cell dynamics across days and state-driven network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show the feasibility of tracking populations of neurons in the same mice, capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

We thank the reviewer for a constructive and positive evaluation of our MS. 

Major points:

(1) The authors used 20 neurons to generate a ground truth dataset. The rationale for this sample size is unclear. Figure 1 indicates the capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

We think this was a misunderstanding of our ground truth dataset analysis which included 192 and not 20 neurons. Indeed, as explained in the methods section, since manually tracking all cells would require prohibitive amounts of time, we decided to generate sparse manual annotations, only tracking a subset of all cells from the first recording day onwards. To do this, we took the first recording (s0), and we defined a grid 64 equidistant points over the FOV and, for each point, identified the closest ROI in terms of euclidean distance from the median pixel of the ROI (see Fig. S3A). We then manually tracked these 64 ROIs across subsequent days. Only neurons that were detected and tracked across all sessions were taken into account and referred to as our ground truth dataset (‘GT’ in Fig. 4). This was done for 3 mice, hence 3X64 neurons and not 20 were used to generate our GT dataset. 

(2) It is unclear how movement was scored in the analysis shown in Figure 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement, or was movement quantified as the amount of time during which the treadmill was displaced?

Movement was scored using a ‘motion energy’ metric as in Stringer et al. 2019 (V1) or Inácio et al. 2025 (S1). This metric takes each two consecutive frames of the videography recordings and computes the difference between them by summing up the square of pixelwise differences between the two images. We made the appropriate changes in the manuscript to further clarify this in the main text and methods in order to avoid confusion.

Since this metric quantifies global movements, it is inherently biased to whole-body movements causing more significant changes in pixel values around the whole FOV of the camera. Slight twitches of a single limb, or the whisker pad would thus contribute much less to this metric, since these are usually slight displacements in a small region of the camera FOV. Additionally, comparing neural activity across all time points (using correlation or R²) also favours movements that last longer (such as wake movements / prolonged periods of high arousal) since each time point is treated equally.

As we suggested in the discussion, in further analysis it would be interesting to look at the link between twitches and neural activity, but this would likely require extensive manual scoring. We could then treat movements not as continuous across all time-points, but instead using event-based analysis for example peri-movement time histograms for different types of movements at different ages, which is however outside of the scope of this study.

(3) The rationale for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

We agree that P11 does not match any change in mouse behavior that we have been able to capture. However, arousal state in mice does change around postnatal day 11. This period marks a transition from immature, fragmented states to more organized and regulated sleep-wake patterns, along with increasing influence from neuromodulatory and sensory systems. All of these changes have been recently reviewed in Wu et al. 2024 (see also Martini et al. 2021). In addition, in the developing somatosensory system, before postnatal day 11 (P11), wake-related movements (reafference) are actively gated and blocked by the external cuneate nucleus (ECN, Tiriac et al. 2016 and all excellent recent work from the Blumberg lab). This gating prevents sensory feedback from wake movements from reaching the cortex, ensuring that only sleep-related twitches drive neural responses. However, around P11, this gating mechanism abruptly lifts, enabling sensory signals from wake movements to influence cortical processing—signaling a dramatic developmental shift from Wu et al. 2024

Reviewer #1 (Recommendations for the authors):

This manuscript represents a significant advancement in the field of developmental neuroscience, offering a powerful and elegant framework for longitudinal cellular tracking using the Track2p method combined with robust analytical approaches. The authors convincingly demonstrate that this integrated methodology provides an invaluable template for investigating complex developmental processes, including the emergence of sensory representations and higher cognitive functions.

A major strength of this work is its emphasis on the power of longitudinal imaging to illuminate activity-dependent development. By tracking the same neurons over time, the authors open up new possibilities to uncover how early activity patterns shape later functional outcomes and the organization of neuronal assemblies-insights that would be inaccessible using conventional cross-sectional designs.

Importantly, the manuscript highlights the potential for this approach to be extended even further, enabling continuous tracking into adulthood and thus offering an unprecedented window into long-term developmental trajectories. The authors also underscore the exciting opportunity to incorporate targeted perturbation experiments, allowing researchers to causally link early circuit dynamics to later outcomes.

Given the increasing recognition that early postnatal alterations can underlie the etiology of various neurodevelopmental disorders, this work is especially timely. The methods and perspectives presented here are poised to catalyze a new generation of developmental studies that can reveal mechanistic underpinnings of both typical and atypical brain development.

In summary, this is a technically impressive and conceptually forward-looking study that sets the stage for transformative advances in developmental neuroscience.

Thank you for the thoughtful feedback—it's greatly appreciated!

Reviewer #2 (Recommendations for the authors):

Minor points:

(1) Figure 1. Consider merging or moving to Supplemental, as its rationale is well described in the text.

We would like to retain the current figure as we believe it provides an effective visual illustration of our rationale that will capture readers' attention and could serve as a valuable reference for others seeking to justify longitudinal tracking of the developing brain. We hope the reviewer will understand our decision.

(2) Some axis labels and panels are difficult to read due to small font sizes (e.g. smaller panels in Figures 5-7).

Modified, thanks 

(3) Supplementary Figures. The order of appearance in the main text is occasionally inconsistent.

This was modified, thanks

(4) Line 132. Add a reference to the registration toolbox used (elastix). A brief description of the affine transformation would also be helpful, either here or in the Methods section (p. 27).

We have added reference to Ntatsis et al. 2023 and described affine transformation in the main text (lines 133-135): 

Firstly, we estimate the spatial transformation between s0 and s1 using affine image registration (i.e. allowing shifting, rotation, scaling and shearing, see Fig. 2B, the transformation is denoted as T).

(5) Lines 147-151. If this method is adapted from another work, please cite the source.

Computing the intersection over union of two ROIs for tracking is a widely established and intuitive method used across numerous studies, representing standard practice rather than requiring specific citation. We have however included the reference to the paper describing the algorithm we use to solve the linear sum assignment problem used for matching neurons across a pair of consecutive days (Crouse 2016).

(6) Line 218. "classical" or automatic?

We meant “classical” in the sense of widely used. 

(7) Lines 220-231. Did the authors find significant variability of successfully tracked neurons across mice? While the data for successfully tracked cells is reported (Figure 5B), the proportions are not. Could differences in neuron dropout across days and mice affect the analysis of neuronal activity statistics?

We thank the reviewer for raising this important point. We computed the fraction of successfully tracked cells in our dataset and found substantial variability:

Cells detected on day 0: [607, 1849, 2190, 1988, 1316, 2138] 

Proportion successfully tracked: [0.47, 0.20, 0.36, 0.37, 0.41, 0.19]

Notably, the number of cells detected on the first day varies considerably (607–2138 cells). There appears to be a trend whereby datasets with fewer initially detected cells show higher tracking success rates, potentially because only highly active cells are identified in these cases.

To draw more definitive conclusions about the proportion of active cells and tracking dropout rates, we would require activity-independent cell detection methods (such as Cellpose applied to isosbestic 830 nm fluorescence, or ideally a pan-neuronal marker in a separate channel, e.g., tdTomato). We have incorporated the tracking success proportions into the revised manuscript.

(8) Line 260. Please briefly explain, here or in the Methods, the rationale for using data from only 3 mice (rather than all 6) for evaluating tracking performance.

We used three mice for this analysis due to the labor-intensive nature of manually annotating 64 ROIs across several days. Given the time constraints of this manual process, we determined that three subjects would provide adequate data to reliably assess tracking performance.

(9) Line 277. Consider clarifying or rephrasing the phrase "across progressively shorter time intervals"? Do you mean across consecutive days?

This has been rephrased as follows: 

Additionally, to assess tracking performance over time, we quantified the proportion of reconstructed ground truth tracks over progressively longer time intervals (first two days, first three days etc. ‘Prop. correct’ in Fig. 4C-F, see Methods). This allowed us to understand how tracking accuracy depends on the number of successive sessions, as well as at which time points the algorithm might fail to successfully track cells.

(10) Line 306. "we also provide additional resources and documentation". Please add a reference or link.

Done, thanks

Track2p  

(11) Lines 342-344. Specify that the raster plots refer to one example mouse, not the entire sample.

Done, thanks.

(12) Lines 996-1002. Please confirm whether only successfully tracked neurons were used to compute the Pearson correlations between all pairs.

Yes of course, this only applies to tracked neurons as it is impossible to compute this for non-tracked pairs.

(13) Line 1003. Add a reference to scikit-learn.

Reference was added to: 

Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V., Vanderplas, J., Passos, A., Cournapeau, D., Brucher, M., Perrot, M., & Duchesnay, E. (2011). Scikit-learn: Machine Learning in Python. Journal of Machine Learning Research, 12, 2825–2830. 

(14) Typos.Correct spacing between numeric values and units.

We did not find many typos regarding spacing between the numerical value and the unit symbol (degrees and percent should not be spaced right?).

Reviewer #3 (Recommendations for the authors):

The font size in many of the figures is too small. For example, it is difficult to follow individual ROIs in Figure S3.

Figure font size has been increased, thanks. In Figure S3 there might have been a misunderstanding, since the three FOV images do not correspond to the FOV of the same mouse across three days but rather to the first recording for each of the three mice used in evaluation (the ROIs can thus not be followed across images since they correspond to a different mouse). To avoid confusion we have labelled each of the FOV images with the corresponding mouse identifier (same as in Fig. 4 and 5).

eLife Assessment

This is a valuable study that explores the role of the conserved transcription factor POU4-2 in the maintenance, regeneration, and function of planarian mechanosensory neurons. The authors present convincing evidence provided by gene expression and functional studies to demonstrate that POU4-2 is required for the maintenance and regeneration of mechanosensory neurons and mechanosensory function in planarians. Furthermore, the authors identify conserved genes associated with human auditory and rheosensory neurons as potential targets of this transcription factor.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors explore the role of the conserved transcription factor POU4-2 in planarian maintenance and regeneration of mechanosensory neurons. The authors explore the role of this transcription factor and identify potential targets of this transcription factor. Importantly, many genes discovered in this work are deeply conserved, with roles in mechanosensation and hearing, indicating that planarians may be a useful model with which to study the roles of these key molecules. This work is important within the field of regenerative neurobiology, but also impactful for those studying evolution of the machinery that is important for human hearing.

Strengths:

The paper is rigorous and thorough, with convincing support for the conclusions of the work.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the role of the transcription factor Smed-pou4-2 in the maintenance, regeneration and function of mechanosensory neurons in the freshwater planarian Schmidtea mediterranea. First, they characterize the expression of pou4-2 in mechanosensory neurons during both homeostasis and regeneration, and examine how its expression is affected by the knockdown of soxB1, 2, a previously identified transcription factor essential for the maintenance and regeneration of these neurons. Second, the authors assess whether pou4-2 is functionally required for the maintenance and regeneration of mechanosensory neurons.

Strengths:

The study provides some new insights into the regulatory role of pou4-2 in the differentiation, maintenance, and regeneration of ciliated mechanosensory neurons in planarians.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review): 

Summary: 

In this manuscript, the authors explore the role of the conserved transcription factor POU4-2 in planarian maintenance and regeneration of mechanosensory neurons. The authors explore the role of this transcription factor and identify potential targets of this transcription factor. Importantly, many genes discovered in this work are deeply conserved, with roles in mechanosensation and hearing, indicating that planarians may be a useful model with which to study the roles of these key molecules. This work is important within the field of regenerative neurobiology, but also impactful for those studying the evolution of the machinery that is important for human hearing. 

Strengths: 

The paper is rigorous and thorough, with convincing support for the conclusions of the work. 

Weaknesses: 

Weaknesses are relatively minor and could be addressed with additional experiments or changes in writing.

Reviewer #2 (Public review): 

Summary: 

In this manuscript, the authors investigate the role of the transcription factor Smed-pou4-2 in the maintenance, regeneration, and function of mechanosensory neurons in the freshwater planarian Schmidtea mediterranea. First, they characterize the expression of pou4-2 in mechanosensory neurons during both homeostasis and regeneration, and examine how its expression is affected by the knockdown of soxB1, 2, a previously identified transcription factor essential for the maintenance and regeneration of these neurons. Second, the authors assess whether pou4-2 is functionally required for the maintenance and regeneration of mechanosensory neurons. 

Strengths: 

The study provides some new insights into the regulatory role of pou4-2 in the differentiation, maintenance, and regeneration of ciliated mechanosensory neurons in planarians. 

Weaknesses: 

The overall scope is relatively limited. The manuscript lacks clear organization, and many of the conclusions would benefit from additional experiments and more rigorous quantification to enhance their strength and impact. 

Reviewing Editor Comments: 

(1) Quantification of pou4-2(+) cells that express (or do not express) hmcn-1-L and/or pkd1L-2(-) is a common suggestion amongst reviewers. It is recognized that Ross et al. (2018) showed that pkd1L-2 and hmcn-1L expression is detected in separate cells by double FISH, and the analysis presented in Supplementary Figure S3 is helpful in showing that some cells expressing pou4-2 (magenta) are not labeled by the combined signal of pkd1L-2 and hmcn-1-L riboprobes (green). However, I am not sure that we can conclude that pkd1L-2 and hmcn-1-L are effectively detected when riboprobes are combined in the analysis. Therefore, quantification of labeled cells as proposed by Reviewers 1 and 2 would help.

Combining riboprobes is a standard approach in the field, and we chose this method as a direct way to determine which cells lack expression of both genes. We agree that providing the raw quantification data would be helpful for readers, and we included this data in Supplementary File S7; the file contains the quantification information for this dFISH experiment represented in Supplementary Figure 3.

(2) It may be helpful to comment on changes (or lack of changes) in atoh gene RNA levels in RNAseq analyses of pou4-2 animals. As mentioned by one of the reviewers, in situs that don't show signal are inconclusive in this regard. 

We fully agree with both reviewers. Two of the planarian atonal homologs are difficult to detect and produce background signals, which we attempted and previously reported in Cowles et al. Development (2013). We conceived performing reciprocal RNAi/in situ experiments, born out of curiosity given the reported role of atonal in the pou4 cascade in other organisms. However, these exploratory experiments lacked a strong rationale for inclusion, particularly given that pou4-2 and the atonal homologs do not share expression patterns, co-expression, or differential expression in our RNA-seq dataset. Therefore, we decided to omit the atonal in situs following pou4-2 RNAi. We retained the experiments showing that knockdown of the atonal genes does not show robust effects on the mechanosensory neuron pattern, as expected. We thank the reviewing editor and reviewers for pinpointing the concern. We agree that additional experiments, such as qPCR experiments, would be needed. We reasoned that while these additional experiments could be informative, they are unlikely to alter the key conclusions of this study substantially.

(3) There seem to be typos at bottom of Figure 10 and top of page 11 when referencing to Figure 4B (should be to 5B instead): "While mechanosensory neuronal patterned expression of Eph1 was downregulated after pou4-2 and soxB1-2 inhibition, low expression in the brain branches of the ventral cephalic ganglia persisted (Figure 4B)." 

Thank you! We have fixed those.

(4) Typo (page 13; kernel?): "...to test to what extent the Pou4 gene regulatory kernel is conserved among these widely divergent animals." 

Regulatory kernels are defined as the minimal sets of interacting genes that drive developmental processes and are the core circuits within a gene regulatory network, but we recognize that this might not be as well known, so we have changed the term to “network” for clarity.

Reviewer #1 (Recommendations for the authors): 

(1) The authors indicate that they are interested in finding out whether POU4-2 is important in the creation of mechanosensory neurons in adulthood as well as in embryogenesis (in other words, whether the mechanism is "reused during adult tissue maintenance and regeneration"). The manuscript clearly shows that planarian POU4 -2 is important in adult neurogenesis in planarians, but there is no evidence presented to show that this is a recapitulation of embryogenesis. Is pou4-2 expressed in the planarian embryo? This might be possible to examine by ISH or through the evaluation of sequencing data that already exists in the literature. 

We agree that these statements should be precise. We have clarified when we make comparisons to the role of Pou4 in sensory system development in other organisms versus its role in the adult planarian. We examined its expression using the existing database of embryonic gene expression. Thanks for hinting at this idea. We performed BLAST in Planosphere (Davies et al., 2017) to cross-reference our clone matching dd_Smed_v6_30562_0_1, which is identical to SMED30002016. The embryonic gene expression for SMED30002016 indicates this gene is expressed at the expected stages given prior knowledge of the timing of organ development in Schmidtea mediterranea (a positive trend begins at Stage 5, with a marked increase by Stage 6 that remains comparable to the asexual expression levels shown). We thank the reviewer for pointing out this oversight. We have incorporated this result in the paper as a Supplementary Figure and discuss how we can only speculate that it has a similar role as we detect in the adult asexual worms.

(2) Can it be determined whether the punctate pou4-2+ cells outside of the stripes are progenitors or other neural cell types? Are there pou4-2+ neurons that are not mechanosensory cell types? Could there be other roles for POU4-2 in the neurogenesis of other cell types? It might help to show percentages of overlap in Figure 4A and discuss whether the two populations add up to 100% of cells. 

These are good questions that arise in part from other statements that need clarification in the text (pointed out by Reviewer 2). We think some of the dorsal pou4-2⁺ might represent progenitor cells undergoing terminal differentiation (see Supplementary Figure 4). We attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. In response to this helpful comment, we have included this question as a future direction in the revised Discussion. Finally, we have edited our description of the expression pattern. We already pointed out that there are other cells on the ventral side that are not affected when soxB1-2 is knocked down. We attempted to resolve the potential identity of those cells working with existing scRNA-seq data in collaboration with colleagues, but their low abundance made it difficult to distinguish other populations. While we acknowledge this interesting possibility, we have chosen to focus this report on the role of pou4-2 downstream of soxB1-2, as this represents the most well-supported aspect of the dataset and was positively highlighted by both the reviewer and editor.

(3) The authors discuss many genes from their analysis that play conserved roles in mechanosensation and hearing. Were there any conserved genes that came up in the analysis of pou4-2(RNAi) planarians that have not yet been studied in human hearing and neurodevelopment? I am wondering the extent to which planarians could be used as a discovery system for mechanosensory neuron function and development, and discussion of this point might increase the impact of this paper or provide critical rationale for expanding work on planarian mechanosensation. 

Indeed, we agree that planarians could be used to identify conserved genes with roles in mechanosensation and have included this point in the Discussion. In this study, we have focused on demonstrating the conservation of gene regulation. While this study was initially based on a graduate thesis project, we have since generated a more comprehensive dataset from isolated heads, which we are currently analyzing. This has been emphasized in the revised Discussion.

Minor: 

(1) For Figure 6E, the authors could consider showing data along a negative axis to indicate a decrease in length in response to vibration and to more clearly show that this decrease doesn't occur as strongly after pou4-2(RNAi). 

We displayed this behavior as the percent change, as this is a standard way to represent this data. As the percent change is a positive value, we represent the data as these positive values.

(2) The authors should consider quantifying the decrease of pou4-2 mRNA after atonal(RNAi) conditions, either by RT-qPCR or cell quantification. Visually, the signal in the stripes after atoh8-2(RNAi) seems lower, particularly in the tail. The punctate pattern outside the stripes may also be decreased after atoh8-1(RNAi). But quantification might strengthen the argument. 

We agree with the reviewer and acknowledge that we should have been more cautious in interpreting these results. Those two genes are difficult to detect and did not show specific patterns in Cowles et al. (2013). The reviewer is correct that additional experiments are necessary before reaching conclusions, but we do not think as discussed earlier we do not think new experiments would provide insights for the major conclusions. These experiments were exploratory in nature and tangential to our main conclusions, especially in the absence of reciprocal evidence (e.g., shared expression patterns, co-expression, or differential expression in our RNA-seq data. Therefore, we decided to eliminate the atonal in situs following pou4-2 RNAi.

Reviewer #2 (Recommendations for the authors): 

A. Expression of pou4-2 in ciliated mechanosensory neurons: 

(1) The conclusion that pou4-2 is expressed in ciliated mechanosensory neurons is primarily based on co-expression analysis using a published single-cell dataset. Although the authors later show that a subset of pou4-2 cells also express pkd1L-2 (Figure 4A), a known marker of ciliated mechanosensory neurons, this finding is not properly quantified. I recommend moving Figure 4A to earlier in the manuscript (e.g., to Figure 2) and expanding the analysis to include additional known markers of this cell type. Proper quantification of the extent of co-localization is necessary to support the claim robustly. 

As pointed out by the reviewer, there is substantive evidence from our lab and other reports. King et al. also showed pou4-2 and pkd1L-2 ‘regulation’ by their scRNA-seq data, and this function is conserved in the acoel Hofstenia miamia (Hulett et al., PNAS 2024 ). Our analysis shows convincing co-localization by scRNA-seq and expression of soxB1-2 and neural markers in the respective populations. Furthermore, we included colocalization of pou4-2 with mechanosensory genes using fluorescence in situ hybridization (Figure 3B, Supplementary Figure 4, and Supplementary File S7). We are confident the data conclusively show pou4-2 regulates pkd1L-2 expression in a subset of mechanosensory neurons. Given the strength of existing observations and previously published data, we believe that additional staining experiments are not essential to support this conclusion. 

(2) There appears to be a conceptual inconsistency in the interpretation of pou4-2 expression dynamics. On one hand, the authors suggest that delayed pou4-2 expression indicates a role in late-stage differentiation (p.6). On the other hand, they propose that pou4-2 may be expressed in undifferentiated progenitors to initiate downstream transcriptional programs (p.8). These interpretations should be reconciled. Additionally, claims regarding pou4-2 expression in progenitor populations should be supported by co-localization with established stem cell or progenitor markers, rather than inferred from signal intensity alone. 

This is an excellent point, and we agree with the reviewer that this section requires editing. As described in response to Reviewer 1, we attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. Furthermore, we could not obtain strong signals in double labeling experiments in pou4-2 in situs combined with piwi-1 or PIWI-1 antibodies. We will include those experiments as a future direction and amend our conclusions accordingly.

(3) The expression pattern shown in Figure 1B raises questions about the precise anatomical localization of pou4-2 cells. It is unclear whether these cells reside in the subepidermal plexus or the deeper submuscular plexus, which represent distinct neuronal layers (Ross et al., 2017). The observed signals near the ventral nerve cords could suggest submuscular localization. To clarify this, higher-resolution imaging and co-staining with region-specific neural markers are recommended. 

In Ross et al. (2018), we showed that the pkd1L-2⁺ cells are located submuscularly. The pkd1L-2 cells express pou4-2, thus the pou4-2⁺ cells are located in the same location. Based on co-expression data and co-expression with PKD genes, we are confident it is submuscular.

B. The functional requirements of pou4-2 in the maintenance of mechanosensory neurons: 

(1) To evaluate the functional role of pou4-2 in maintaining mechanosensory neurons, the authors performed whole-animal RNA-seq on pou4-2(RNAi) and control animals, identifying a significant downregulation of genes associated with mechanosensory neuron expression. However, the presentation of these findings is fragmented across Figures 3, 4, and 5. I recommend consolidating the RNA-seq results (Figure 3) and the subsequent validation of downregulated genes (Figures 4 and 5) into a single, cohesive figure. This would improve the logical flow and clarity of the manuscript. 

As suggested by the reviewer, we have combined Figures 3 and 4 (new Figure 3), which we believe improves the flow. We decided to keep Figure 5 (new Figure 4) as a standalone because it focuses on the characterization of new genes revealed by RNAseq and scRNA-seq data mining that were not previously reported in Ross et al. 2018 and

2024.

(2) In pou4-2(RNAi) animals, pkd1L-2 expression appears to be entirely lost, while hmcn-1-L shows faint expression in scattered peripheral regions. The authors suggest that an extended RNAi treatment might be necessary to fully eliminate hmcn-1-L expression. However, an alternative explanation is that pou4-2 is not essential for maintaining all hmcn-1-L cells, particularly if pou4-2 expression does not fully overlap with that of hmcn-1-L. This possibility should be acknowledged and discussed. 

We agree and have acknowledged this point in the revised text.

(3) On page 9, the section title claims that "Smed-pou4-2 regulates genes involved in ciliated cell structure organization, cell adhesion, and nervous system development." While some differentially expressed genes are indeed annotated with these functions based on homology, the manuscript does not provide experimental evidence supporting their roles in these biological processes in planarians. The title should be revised to avoid overstatement, and the limitations of extrapolating a function solely from gene annotation should be acknowledged. 

Excellent point. We have edited the text to indicate that the genes were annotated or implicated.

(4) The cilia staining presented in Figure 6B to support the claim that pou4-2 is required for ciliated cell structure organization is unconvincing. Improved imaging and more targeted analysis (e.g., co-labeling with mechanosensory markers) are needed to support this conclusion. 

We have addressed this concern by adjusting the language to be more precise and indicate that the stereotypical banded pattern is disrupted with decreased cilia labeling along the dorsal ciliated stripe. Indeed, our conclusion overstated the observations made with the staining and imaging resolution. Thank you.

C. The functional requirements of pou4-2 in the regeneration of mechanosensory neurons: 

To evaluate the role of pou4-2 in the regeneration of mechanosensory neurons, the authors performed amputations on pou4-2(RNAi) and control(RNAi) animals and assessed the expression of mechanosensory markers (pkd1L-2, hmcn-1-L) alongside a functional assay. However, the results shown in Figure 4B indicate the presence of numerous pkd1L-2 and hmcn-1-L cells in the blastema of pou4-2(RNAi) animals. This observation raises the possibility that pou4-2 may not be essential for the regeneration of these mechanosensory neurons. The authors should address this alternative interpretation. 

Our interpretation is that there were very few cells expressing the markers compared to controls. The pattern was predominantly lost, which is consistent with other experiments shown in the paper. However, we have added the additional caveat suggested by the reviewer.

Minor points: 

(1) On p.8, the authors wrote "every 12 hours post-irradiation". However, this is not consistent with the figure, which only shows 0, 3, 4, 4.5, 5, and 5.5 dpi. 

We corrected this. Thank you for catching the mistake!

(2) On p.12, the authors wrote "Analysis of pou4-2 RNAi data revealed differentially expressed genes with known roles in mechanosensory functions, such as loxhd-1, cdh23, and myo7a. Mutations in these genes can cause a loss of mechanosensation/transduction". This is misleading because, to my knowledge, the role of these genes in planarians is unknown. If the authors meant other model systems, they should clearly state this in the text and include proper references. 

The reviewer is correct that we are referencing findings from other organisms. We have clarified this point in the revised text. The appropriate references were included and cited in the first version.

(3) On p.7, the authors wrote, "conversely, the expression of atonal genes was unaffected in pou4-2 RNAi-treated regenerates (Supplementary Figure S2B)". However, it is unclear whether the Atoh8-1 and Atoh8-2 signals are real, as the quality of the in situ results is too low to distinguish between real signals and background noise/non-specific staining. 

This valid concern was addressed in our response to Reviewer 1. We have adjusted the figure and the text accordingly.

(4) On p.6 the authors wrote "pinpointed time points wherein the pou4-2 transcripts were robustly downregulated". However, the current version of the manuscript does not provide data explaining why Pou4-2 transcripts are robustly downregulated on day 12. 

Yes, we determined the appropriate time points using qPCR for all sample extractions. As an example, see the figure for qPCR validation at day 12 showing that pou4-2 and pkd1L2 are down.

Author response image 1.

In this graph, samples labeled “G” represent four biological controls of gfp(RNAi) control animals, and samples labeled “P” represent four biological controls of pou4-2(RNAi)animals at day 12 in the RNAi protocol.

(5) On p.13, the authors wrote "collecting RNA from how animals." Is this a typo? 

Thanks for catching the typo. It should read “whole” animals. We have corrected this.

(6) On p.14, the authors wrote "but the expression patterns of planarian atonal genes indicated that they represent completely different cell populations from pou4-2-regulated mechanosensory neurons". However, this is unclear from the images, as the in situ staining of Atoh8-1 and Atoh82 are potentially failed stainings. 

We agree. We have edited accordingly.

eLife Assessment

This valuable manuscript presents an open-source and low-cost acoustic system for quantifying biting and chewing in mice. The approach is carefully validated against human observers, demonstrating strong methodological reliability and enabling high-resolution analysis of feeding microstructure. The tool has broad relevance for studies of appetite circuits and pharmacological interventions. A significant contribution is the identification of previously unrecognized "meal-related" neurons in the lateral hypothalamus, providing novel biological insight into food consumption. While the support for the methodological advances is compelling and robust, some circuit-level conclusions are preliminary or incomplete, relying on small pilot samples and manual classification, and should be interpreted with caution. This paper will be of interest to those interested in ingestive behavior and/or hypothalamus.

Reviewer #1 (Public review):

This is an interesting and valuable paper by Gil-Lievana, Arroyo et al. that presents an open-source method (the "Crunchometer") for quantifying biting and chewing behavior in mice using audio detection. The work addresses an important and unmet need in the field: quantitative measures of feeding behavior with solid foods, since most prior approaches have been limited to liquids. The authors make a clear and compelling case for why this problem is important, and I fully agree with their motivation.

The system is carefully validated against human-scored video data and is shown to be at least as accurate, and in some cases more accurate, than human observers. This is a major strength of the study. I also particularly appreciate the demonstration of the technology in the context of LHA circuitry, which nicely illustrates its utility and importance for mechanistic studies of feeding. I also appreciate the ability to readily time-lock neural data to individual crunches. Overall, the manuscript is well-executed and represents a useful contribution to the field.

The comments I have are largely minor and should be straightforward to address:

(1) The authors should report sample sizes for all mouse cohorts, either alongside the statistics or in the figure legends for mean data.

(2) Clarification is needed as to whether crunch detection fidelity is influenced by the hardness or softness of the food. The focus here is on standard pellets, with some additional high-fat pellet data, but it would be useful to know how generalizable the method is across different textures.

(3) The authors should comment on how susceptible the Crunchometer is to background noise. For example, how well does it perform in the presence of white noise, experimenter movement, or other task-related sounds?

(4) Chemogenetic activation of LHA GABAergic neurons is used. DREADD-based activation may strongly drive these neurons in a way that is not directly comparable to optogenetic or more physiological manipulations. While I do not think additional experiments are required, it would strengthen the discussion to briefly acknowledge this limitation.

Reviewer #2 (Public review):

Summary:

This manuscript introduces the Crunchometer, a low-cost, open-source acoustic platform for monitoring the microstructure of solid food intake in mice. The Crunchometer is designed to overcome the limitations of existing methods for studying feeding behavior in rodents. The goal was to provide a tool that could precisely capture the microstructure of solid food intake, something often overlooked in favor of liquid-based assays, while being affordable, scalable, and compatible with neural recording techniques. By doing so, the authors aimed to enable detailed analysis of how physiological states, drugs, and specific neural circuits shape naturalistic feeding behaviors.

Strengths:

The study's strengths lie in its clear innovation, methodological rigor in validation against human annotation, and demonstration of broad utility across behavioral and neuroscience paradigms. The approach addresses a significant methodological gap in the field by moving beyond liquid-based feeding assays and provides an accessible tool for precisely dissecting ingestive behavior. The system is validated across multiple contexts, including physiological state (fed vs. fasted), pharmacological manipulation (semaglutide), and circuit-level interventions (chemogenetic activation of LH neurons), and is further shown to integrate seamlessly with both electrophysiology and calcium imaging.

(1) Introduces a low-cost, open-source acoustic tool for measuring solid food intake, filling a critical gap left by expensive and proprietary systems.

(2) Makes the method easily adoptable across labs with detailed setup instructions and shared benchmark datasets.

(3) Provides high temporal precision for detecting bite events compared to human observers.

(4) Successfully distinguishes feeding microstructure (bites, bouts, IBIs, gnawing vs. consumption) with greater objectivity than manual annotation.

(5) Demonstrates compatibility with electrophysiology and calcium imaging, enabling fine-scale alignment of neural activity with feeding behavior.

(6) Effectively discriminates between fed vs. fasted states, validating physiological sensitivity.

(7) Captures the pharmacological effects of semaglutide, although this is really just reduced feeding and associated readouts (bouts, latency, etc).

(8) Has potential to distinguish consummatory vs. non-consummatory behaviors (e.g., food spillage, gnawing); however, the current SVM model struggles to separate biting from gnawing due to similar acoustic profiles, and manual validation is still required.

(9) Provides potential for closed-loop experiments.

Weaknesses:

Several limitations temper the strength of the conclusions: the supervised classifier still requires manual correction for gnawing, generalizability across different setups is limited, and the neuroscience findings, particularly calcium imaging of GABAergic and glutamatergic neurons, are based on small pilot samples. These issues do not undermine the value of the tool, but mean that the neural circuit findings should be interpreted as preliminary.

(1) Some neuroscience findings (calcium imaging of GABAergic vs. glutamatergic neurons) are based on small pilot samples (n=2 mice per condition), limiting generalizability.

(2) Chemogenetic and pharmacological experiments used small cohorts, raising statistical power concerns.

(3) Correlation with actual food intake is modest and sometimes less accurate than human observers.

(4) Sensitive to hoarding behavior, which can reduce detection accuracy and requires manual correction for misclassifications (e.g., tail movements, non-food noises). However, these limitations are discussed and not ignored.

Conclusion:

Overall, this is an exciting and impactful methodological advance that will likely be widely adopted in the field. I recommend minor revisions to clarify the limits of classifier generalizability, better contextualize the small-sample neuroscience findings as pilot data, and discuss future directions (e.g., real-time closed-loop applications).

Reviewer #3 (Public review):

Summary:

The manuscript provides detailed information on the construction of open-source systems to monitor ingestive behavior with low-cost equipment. Overall, this is a welcome addition to the arsenal of equipment that could be used to make measurements. The authors show interesting applications with data that reveal important neurophysiological properties of neurons in the lateral hypothalamus. The identification of previously unknown "meal-related" neurons in the LH highlights the utility of the device and is a novel insight that should spark further investigation on the LH. This manuscript and videos provide a wealth of useful information that should be a must-read for anyone in the ingestive behavior or hypothalamus fields.

A scholarly introduction to the history and utility of various ways feeding is measured in rodents is provided. One point - the microstructure of eating solid food - has been studied extensively (for one of many studies, see https://doi.org/10.1371/journal.pone.0246569 ). However, I agree that the crunchometer will allow for more people to access recordings during food intake and temporally lock consummatory behavior to neural activity.

Questions on results:

(1) It is unclear why 10% sucrose solution was used as a liquid instead of water, given that the study is focusing on the solid food source.

(2) It is unclear how essential the human verification is in the pipeline - results for Figure 1 keep referring to the verification as essential. Is that dispensable once the ML algorithms have been trained?

(3) The ability to extrapolate food quantity consumed is limited, with high variability. This limitation does not undercut the utility of the crunchometer, but should be highlighted as one of the parameters that are not suitable for this system. This limitation should be added to the limitations section.

(4) The ability to discriminate between gnawing and consummatory behavior is a strength (Figure 5), and these findings are important. However, it is unclear what can be made of mice that have 'gnawing' behavior in the fasted state (like in Figure 3). It seems they would need to be eliminated from the analysis with this tool?

(5) Why is there a post-semaglutide fed group and not a fasted group in Figure 4? It seems both would have been interesting, as one could expect an effect on feeding even 24h after semaglutide treatment. This would help parse the preference better because the animals eat such a small amount on semaglutide, that it is hard to compare to the fasted condition with saline treatment.

(6) The identification of 'meal-related' neurons in the LH is another strength of the manuscript. Although there is currently insufficient data, could similar recordings be used to give a neurophysiological definition of a 'meal' duration/size? Typically, these were somewhat arbitrarily defined behaviorally. Having a neural correlate to a 'meal' would be a powerful tool for understanding how meals are involved in overall caloric intake.

(7) The conclusion in the title of Figure 8 is premature, given the pilot nature and small number of neurons and mice sampled.

Conclusion:

Overall, this report on the Crunchometer is well done and provides a valuable tool for all who study food intake and the behaviors around food intake. Clarification or answers to the points above will only further the utility and understanding of the tool for the research community. I am excited to see the future utility of this tool in emerging research.

Yes - the oral history project incorporated the 5Rs. I particularly think the "reach" element was present. By recording the interviews and sharing them on a website - it allows students in class (and beyond) to hear these stories.

Yes - definitely. The oral history project helped to share stories of people and their lived experiences. This was an innovative way to share those stories with a broader audience. I enjoyed (and learned) from listening to some.

I really liked the Oral History project that was used in the HIST 150 class at JMU. I enjoyed listening to some of the stories and reminded me of NPR's Story Corps. I can see using something like this in my Population Health Determinants Class to capture student reflections from their service-learning experiences.

I’m thinking about how open pedagogy connects really naturally with some of the things I already do in my classes. In Population Health Determinants, for example, there’s a service-learning component where students engage with local organizations to understand social determinants of health. I can see opportunities for students to create open educational materials or community resources from those experiences—something that lives beyond the course and benefits others.

Similarly, in U.S. and Global Health Care Systems, my students do a podcast project comparing different countries’ health systems. That assignment already emphasizes collaboration and knowledge sharing, but I’d like to take it further by having them publish their work openly so future students (or even community members) can learn from it.

eLife Assessment

This paper is an important overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, providing a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects. The overall synthesis is convincing. The database proposed by the paper has the potential to become a key community resource if carefully curated and developed.

Reviewer #1 (Public review):

This paper is a relevant overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems, nevertheless, relevant to summarise the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data free permutation of the parameter space.

A database summarising the literature and allowing for quantitative assessment of these studies is a key contribution of the paper. If curated well, it can become a valuable community resource.

Comments on revisions:

The paper is much improved. There remain a few caveats the authors may want to address.

I'm not going to dwell on this if the authors don't agree, but remain critical about the inclusion of TPS in the discussion. It's comparing apples and oranges, and unless there's a personal interest the authors have in TPS, it remains puzzling why it is included in the first place. As per my previous review, the literature on TPS, and especially the main example cited, has been highly criticised, including national patient and medical associations. A mere disclaimer that more work is needed isn't enough, in this reviewer's opinion - I simply don't understand why the authors go out on a limb here when the rest of the paper is done so well and thoroughly.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

This paper is a relevant overview of the currently published literature on lowintensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems nevertheless relevant to summarize the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data-free permutation of the parameter space.

The meta-analysis concludes that there are, at best, weak trends toward specific parameters predicting the direction of the stimulation effects. The data have been incorporated into an open database that will ideally continue to be populated by the community and thereby become a helpful resource as the field moves forward.

Strengths:

The current state of human TUS is concisely and well summarized. The methods of the meta-analysis are appropriate. The database is a valuable resource.

We thank the reviewer for their positive assessment of the revised manuscript and the potential importance of the resource to the TUS community. 

Suggestions:

The paper remains lengthy and somewhat unfocused, to the detriment of readability. One can understand that the authors wish to include as much information as possible, but this reviewer is sceptical that this will aid the use of the databank, or help broaden the readership. For one, there is a good chunk of repetition throughout. The intro is also somewhat oscillating between TMS, tDCS and TUS. While the former two help contextualizing the issue, it doesn't seem necessary. In the section on clinical applications of TUs and possible outcomes of TUS, there's an imbalance of the content across examples. That's in part because of the difference in knowledge base but some sections could probably be shortened, eg stroke. In any case, the authors may want to consider whether it is worth making some additional effort in pruning the paper

We thank the reviewer for these suggestions. We have checked for redundancy and that the clinical review section is more balanced, although some of the sections have more TUS studies than others, therefore some imbalance is unavoidable. As some examples, we have condensed the “Stroke and neuroprotection in brain injury” section (lines 624-647). This helps to improve the clarity and readability of the manuscript.

The terms or concept of enhancement and suppression warrant a clearer definition and usage. In most cases, the authors refer to E/S of neural activity. Perhaps using terms such as "neural enhancement" etc helps distinguish these from eg behavioural or clinical effects. Crucially, how one maps onto the other is not clear. But in any case, a clear statement that the changes outlined on lines 277ff do not

We thank the reviewer for this point and agree that it is important to distinguish neural E/S, as we had intended, from behavioral effects. In the first instance and in several places we add ‘neural’ before enhancement/suppression.  Also see Lines 276-279: “Probable net neural enhancement versus suppression was characterised as follows. Note that our use of the terms enhancement and suppression refers exclusively to the increase or decrease of neural activity, respectively, as measured by, neurophysiological methods (EEG-ERPs, BOLD fMRI, etc.) and does not imply equivalent changes in behavioural responses” 

Please see also lines 108-116.

Re tb-TUS (lines 382ff), it is worth acknowledging here that independent replication is very limited (eg Bao et al 2024; Fong et al bioRxiv 2024) and seems to indicate rather different effects

We have updated this section by referencing Bao et al. and Fong et al., as examples of the limited independent replication of tbTUS results. Please see lines 392-396. “However, independent replication of these findings remains limited. For example, Bao, found reduced motor cortex excitability – measured as decreased TMS-MEP amplitude in M1 -- that lasted up to 30 minutes post-sonication (Bao et al., 2024). Whereas Fong reported no significant effects between tbTUS and sham conditions in M1 excitability (Fong et al., 2024).”

The comparison with TPS is troublesome. For one, that original study was incredibly poorly controlled and designed. Cherry-picking individual (badly conducted) proof-of-principle studies doesn't seem a great way to go about as one can find a match for any desired use or outcome. Moreover, other than the concept of "pulsed" stimulation, it is not clear why that original study would motivate the use of TUS in the way the authors propose; both types of stimulation act in very different ways (if TPS "acts" at all). But surely the cited TPS study does not "demonstrate the capability for TUS for pre-operative cognitive mapping". As an aside, why the authors feel the need to state the "potential for TPS... to enhance cognitive function" is unclear, but it is certainly a non-sequitur. This review feels quite strongly that simplistic analogies such as the one here are unnecessary and misleading, and don't reflect the thoughtful discussion of the rest of the paper. In the other clinical examples, the authors build their suggestions on other TUS studies, which seems more sensible.

This is an excellent point, and we have removed that statement replacing it with: “However, TPS effects studies remain highly limited and would require further study and comparison to effects with other TUS protocols.”. Please see lines 561-562. We thank the reviewer for the supportive comments on the rest of the review.

Notice that the way the equation solves that with the solved value. If 10^pH-pKA is greater than 1, the Conjugate Base or [A-] is in higher concentration than the lewis acid [HA], alternatively an increased HA concentration means a larger denominator --> 10^pH-pKA closer to 0; Conjugate Acid is higher in concentration.

In order to obtain a buffer, the complete neutralization of either the acid or the base must be avoided. Thus weak acids are used (and my guess no strong bases as well) because the complete dissociation of its ions would prevent the suspension of the pH.

Why is it tending so much towards yes (before becoming uncertain)? Maybe the model tends to favor positive responses, as mentioned earlier being the case for another model. But I hypothesized there to be relation hallucinations with the yes and no flipped compared to the Figure. Since the plot is averaged, are those cases so rare that they do not get represented after averaging anymore?

Why does it become uncertain only once the last layers are reached? They call it sharp change in Figure 8b.

That assumes that the token that has high probability in the intermediate layer (e.g. yes in Figure 7) is the correct answer.

This amount isn’t correct. We need to find a way to get the exact or a near-exact figure for that.

eLife Assessment

This important study addresses a topic that is frequently discussed in the literature but is under-assessed, namely correlations among genome size, repeat content, and pathogenicity in fungi. Contrary to previous assertions, the authors found that repeat content is not associated with pathogenicity. Rather, pathogenic lifestyle was found to be better explained by the number of protein-coding genes, with other genomic features associated with insect association status. The results are considered solid, although there remain concerns about potential biases stemming from the underlying data quality of the analyzed genomes.

Reviewer #1 (Public review):

Summary:

The manuscript "Lifestyles shape genome size and gene content in fungal pathogens" by Fijarczyk et al. presents a comprehensive analyses of a large dataset of fungal genomes to investigate what genomic features correlate with pathogenicity and insect associations. The authors focus on a single class of fungi, due to the diversity of life styles and availability of genomes. They analyze a set of 12 genomic features for correlations with either pathogenicity or insect association and find that, contrary to previous assertions, repeat content does not associate with pathogenicity. They discover that the number of protein coding genes, including total size of non-repetitive DNA does correlate with pathogenicity. However, unique features are associated to insect associations. This work represents an important contribution to the attempts to understand what features of genomic architecture impact the evolution of pathogenicity in fungi.

Strengths:

The statistical methods appear to be properly employed and analyses thoroughly conducted. The size of the dataset is impressive and likely makes the conclusions robust. The manuscript is well written and the information, while dense, is generally presented in a clear manner.

Weaknesses:

My main concerns all involve the genomic data, how they were annotated, and the biases this could impart to the downstream analyses. The three main features I'm concerned with are sequencing technology, gene annotation, and repeat annotation. The authors have done an excellent investigation into these issues, but these show concerning trends, and my concerns are not as assuaged as the authors.

The collection of genomes is diverse and includes assemblies generated from multiple sequencing technologies including both short- and long-read technologies. From the number of scaffolds its clear that the quality of the assemblies varies dramatically, even within categories of long- and short-read. This is going to impact many of the values important for this study, as the authors show.

I have considerable worries that the gene annotation methods could impart biases that significantly effect the main conclusions. Only 5 reference training sets were used for the Sordariomycetes and these are unequally distributed across the phylogeny. Augusts obviously performed less than ideally, as the authors observe in their extended analysis. While the authors are not concerned about phylogenetic distance from the training species, due to prevailing trends, I am not as convinced. In figure S12, the Augustus features appear to have considerably more variation in values for the H2 set and possible the microascales. It is unclear how this would effect the conclusions in this study.

Unfortunately, the genomes available from NCBI will vary greatly in the quality of their repeat masking. While some will have been masked using custom libraries generated with software like Repeatmodeler, others will probably have been masked with public databases like repbase. As public databases are again biased towards certain species (Fusarium is well represented in repbase for example), this could have significant impacts on estimating repeat content. Additionally, even custom libraries can be problematic as some software (like RepeatModeler) will included multicopy host genes leading to bona fide genes being masked if proper filtering is not employed. A more consistent repeat masking pipeline would add to the robustness of the conclusions. The authors show that there is a significant bias in their set.

To a lesser degree I wonder what impact the use of representative genomes for a species has on the analyses. Some species vary greatly in genome size, repeat content and architecture among strains. I understand that it is difficult to address in this type of analysis, but it could be discussed.

Reviewer #2 (Public review):

Summary:

In this paper, the authors report on the genomic correlates of the transition to the pathogenic lifestyle in Sordariomycetes. The pathogenic lifestyle was found to be better explained by the number of genes, and in particular effectors and tRNAs, but this was modulated by the type of interacting host (insect or not insect) and the ability to be vectored by insects.

Strengths:

The main strengths of this study lie in (i) the size of the dataset, and the potentially high number of lifestyle transitions in Sordariomycetes, (ii) the quality of the analyses and the quality of the presentation of the results, (iii) the importance of the authors' findings.

Weaknesses:

The weakness is a common issue in most comparative genomics studies in fungi, but it remains important and valid to highlight it. Defining lifestyles is complex because many fungi go through different lifestyles during their life cycles (for instance, symbiotic phases interspersed with saprotrophic phases). In many fungi, the lifestyle referenced in the literature is merely the sampling substrate (such as wood or dung), which does not necessarily mean that this substrate is a key part of the life cycle. The authors discuss this issue, but they do not eliminate the underlying uncertainties.

Author response:

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

Reviewer #1 (Public review):

Summary:

The manuscript "Lifestyles shape genome size and gene content in fungal pathogens" by Fijarczyk et al. presents a comprehensive analysis of a large dataset of fungal genomes to investigate what genomic features correlate with pathogenicity and insect associations. The authors focus on a single class of fungi, due to the diversity of lifestyles and availability of genomes. They analyze a set of 12 genomic features for correlations with either pathogenicity or insect association and find that, contrary to previous assertions, repeat content does not associate with pathogenicity. They discover that the number of proteincoding genes, including the total size of non-repetitive DNA does correlate with pathogenicity. However, unique features are associated with insect associations. This work represents an important contribution to the attempts to understand what features of genomic architecture impact the evolution of pathogenicity in fungi.

Strengths:

The statistical methods appear to be properly employed and analyses thoroughly conducted. The manuscript is well written and the information, while dense, is generally presented in a clear manner.

Weaknesses:

My main concerns all involve the genomic data, how they were annotated, and the biases this could impart to the downstream analyses. The three main features I'm concerned with are sequencing technology, gene annotation, and repeat annotation.

We thank the reviewer for all the comments. We are aware that the genome assemblies are of heterogeneous quality since they come from many sources. The goal of this study was to make the best use of the existing assemblies, with the assumption that noise introduced by the heterogeneity of sequencing methods should be overcome by the robustness of evolutionary trends and the breadth and number of analyzed assemblies. Therefore, at worst, we would expect a decrease in the power to detect existing trends. It is important to note that the only way to confidently remove all potential biases would be to sequence and analyze all species in the same way; this would require a complete study and is beyond the scope of the work presented here. Nevertheless some biases could affect the results in a negative way, eg. is if they affect fungal lifestyles differently. We therefore made an attempt to explore the impact of sequencing technology, gene and repeat annotation approach among genomes of different fungal lifestyles. Details are described in Supplementary Results and below. Overall, even though the assembly size and annotations conducted with Augustus can sometimes vary compared to annotations from other resources, such as JGI Mycocosm, we do not observe a bias associated with fungal lifestyles. Comparison of annotations conducted with Augustus and JGI Mycocosm dataset revealed variation in gene-related features that reflect biological differences rather than issues with annotation.  

The collection of genomes is diverse and includes assemblies generated from multiple sequencing technologies including both short- and long-read technologies. Not only has the impact of the sequencing method not been evaluated, but the technology is not even listed in Table S1. From the number of scaffolds it is clear that the quality of the assemblies varies dramatically. This is going to impact many of the values important for this study, including genome size, repeat content, and gene number.

We have now added sequencing technology in Table S1 as it was reported in NCBI. We evaluated the impact of long-read (Nanopore, PacBio, Sanger) vs short-read assemblies in Supplementary Results. In short, the proportion of different lifestyles (pathogenic vs. nonpathogenic, IA vs non-IA) were the same for short- and long-read assemblies. Indeed, longread assemblies were longer, had a higher fraction of repeats and less genes on average, but the differences between pathogenic vs. non-pathogenic (or IA vs non-IA) species were in the same direction for two sequencing technologies and in line with our results. There were some discrepancies, eg. mean intron length was longer for pathogens with long-read assemblies, but slightly shorter on average for short-read assemblies (and to lesser extent GC and pseudo tRNA count), which could explain weaker or mixed results in our study for these features.

Additionally, since some filtering was employed for small contigs, this could also bias the results.

The reason behind setting the lower contig length threshold was the fact that assemblies submitted to NCBI have varying lower-length thresholds. This is because assemblers do not output contigs above a certain length, and this threshold can be manipulated by the user. Setting a common min contig length was meant to remove this variation, knowing that any length cut-off will have a larger effect on short-read based assemblies than long-read-based assemblies. Notably, genome assemblies of corresponding species in JGI Mycocosm have a minimum contig length of 865 bp, not much lower than in our dataset. Importantly, in a response to a comment of previous reviewer, repeat content was recalculated on raw assembly lengths instead of on filtered assembly length. 

I have considerable worries that the gene annotation methods could impart biases that significantly affect the main conclusions. Only 5 reference training sets were used for the Sordariomycetes and these are unequally distributed across the phylogeny. Augusts obviously performed less than ideally, as the authors reported that it under-annotated the genomes by 10%. I suspect it will have performed worse with increasing phylogenetic distance from the reference genomes. None of the species used for training were insectassociated, except for those generated by the authors for this study. As this feature was used to split the data it could impact the results. Some major results rely explicitly on having good gene annotations, like exon length, adding to these concerns. Looking manually at Table S1 at Ophiostoma, it does seem to be a general trend that the genomes annotated with Magnaporthe grisea have shorter exons than those annotated with H294. I also wonder if many of the trends evident in Figure 5 are also the result of these biases. Clades H1 and G each contain a species used in the training and have an increase in genes for example.

We have applied 6 different reference training sets (instead of one) precisely to address the problem of increasing phylogenetic distance of annotated species. To further investigate the impact of chosen species for training, we plotted five gene features (number of genes, number of introns, intron length, exon length, fraction of genes with introns) as a function of   branch length distance from the species (or genus) used as a training set for annotation. We don’t see systematic biases across different training sets. However,  trends are very clear for clades annotated with fusarium. This set of species includes Hypocreales and Microascales, which is indeed unfortunate since Microascales is an IA group and at the same time the most distant from the fusarium genus in this set. To clarify if this trend is related to annotation bias or a biological trend, we compared gene annotations with those of Mycocosm, between Hypocreales Fusarium species, Hypocreales non-Fusarium species, and Microascales, and we observe exactly the same trends in all gene features. 

Similarly, among species that were annotated with magnaporthe_grisea, Ophiostomatales (another IA group) are among the most distant from the training set species. Here, however, another order, Diaporthales, is similarly distant, yet the two orders display different feature ranges. In terms of exon length, top 2 species in this training set include Ophiostoma, and they reach similar exon length as the Ophiostoma species annotated using H294 as a training set. In summary, it is possible that the choice of annotation species has some effect on feature values; however, in this dataset, these biases are likely mitigated by biological differences among lifestyles and clades. 

Unfortunately, the genomes available from NCBI will vary greatly in the quality of their repeat masking. While some will have been masked using custom libraries generated with software like Repeatmodeler, others will probably have been masked with public databases like repbase. As public databases are again biased towards certain species (Fusarium is well represented in repbase for example), this could have significant impacts on estimating repeat content. Additionally, even custom libraries can be problematic as some software (like RepeatModeler) will include multicopy host genes leading to bona fide genes being masked if proper filtering is not employed. A more consistent repeat masking pipeline would add to the robustness of the conclusions.

We have searched for the same species in JGI Mycocosm and were able to retrieve 58 genome assemblies with matching species, with 19 of them belonging to the same strain as in our dataset. Overall we found no differences in genome assembly length. Interestingly, repeat content was slightly higher for NCBI genome assemblies compared to JGI Mycocosm assemblies, perhaps due to masking of host multicopy genes, as the reviewer mentioned. By comparing pathogenic and non-pathogenic species for the same 19 strains, we observe that JGI Mycocosm annotates fewer repeats in pathogenic species than Augustus annotations (but trends are similar when taking into account 58 matching species). Given a small number of samples, it is hard to draw any strong conclusions; however, the differences that we see are in favor of our general results showing no (or negative) correlation of repeat content with pathogenicity. 

To a lesser degree, I wonder what impact the use of representative genomes for a species has on the analyses. Some species vary greatly in genome size, repeat content, and architecture among strains. I understand that it is difficult to address in this type of analysis, but it could be discussed.

In our case the use of protein sequences could underestimate divergence between closely related strains from the same species. We also excluded strains of the same species to avoid overrepresentation of closely related strains with similar lifestyle traits. We agree that some changes in the genome architecture can occur very rapidly, even at the species level, though analyzing emergence of eg. pathogenicity at the population level would require a slightly different approach which accounts for population-level processes. 

Reviewer #2 (Public review):

Summary:

In this paper, the authors report on the genomic correlates of the transition to the pathogenic lifestyle in Sordariomycetes. The pathogenic lifestyle was found to be better explained by the number of genes, and in particular effectors and tRNAs, but this was modulated by the type of interacting host (insect or not insect) and the ability to be vectored by insects.

Strengths:

The main strength of this study lies in the size of the dataset, and the potentially high number of lifestyle transitions in Sordariomycetes.

Weaknesses:

The main strength of the study is not the clarity of the conclusions.

(1) This is due firstly to the presentation of the hypotheses. The introduction is poorly structured and contradictory in some places. It is also incomplete since, for example, fungusinsect associations are not mentioned in the introduction even though they are explicitly considered in the analyses.

We thank the reviewer for pointing this out. We strived to address all comments and suggestions of the reviewer to clarify the message and remove the contradictions. We also added information about why we included insect-association trait in our analysis. 

(2) The lack of clarity also stems from certain biases that are challenging to control in microbial comparative genomics. Indeed, defining lifestyles is complicated because many fungi exhibit different lifestyles throughout their life cycles (for instance, symbiotic phases interspersed with saprotrophic phases). In numerous fungi, the lifestyle referenced in the literature is merely the sampling substrate (such as wood or dung), which doesn't mean that this substrate is a crucial aspect of the life cycle. This issue is discussed by the authors, but they do not eliminate the underlying uncertainties.

We agree with the reviewer that lack of certainty in the lifestyle or range of possible lifestyles of studied species is a weakness in this analysis. We are limited by the information available in the literature. We hope that our study will increase interest in collecting such data in the future.

Reviewer #3 (Public review):

Summary:

This important study combines comparative genomics with other validation methods to identify the factors that mediate genome size evolution in Sordariomycetes fungi and their relationship with lifestyle. The study provides insights into genome architecture traits in this Ascomycete group, finding that, rather than transposons, the size of their genomes is often influenced by gene gain and loss. With an excellent dataset and robust statistical support, this work contributes valuable insights into genome size evolution in Sordariomycetes, a topic of interest to both the biological and bioinformatics communities.

Strengths:

This study is complete and well-structured.

Bioinformatics analysis is always backed by good sampling and statistical methods. Also, the graphic part is intuitive and complementary to the text.

Weaknesses:

The work is great in general, I just had issues with the Figure 1B interpretation.

I struggled a bit to find the correspondence between this sentence: "Most genomic features were correlated with genome size and with each other, with the strongest positive correlation observed between the size of the assembly excluding repeats and the number of genes (Figure 1B)." and the Figure 1B. Perhaps highlighting the key p values in the figure could help.

We thank the reviewer for pointing out this sentence. Perhaps the misunderstanding comes from the fact that in this sentence one variable is missing. The correct version should be “Most genomic features were correlated with genome size and with each other, with the strongest positive correlation observed between the genome size, the genome size excluding repeats and the number of genes (Figure 1B)”. Also, the variable names now correspond better to those shown on the figure.

Reviewer #1 (Recommendations for the authors):

The authors have clearly done a lot of good work, and I think this study is worthwhile. I understand that my concerns about the underlying data could necessitate rerunning the entire analysis with better gene models, but there may be another option. JGI has a fairly standard pipeline for gene and repeat annotation. Their gene predictions are based on RNA data from the sequenced strain and should be quite good in general. One could either compare the annotations from this manuscript to those in mycocosm for genomes that are identical and see if there are systematic biases, or rerun some analyses on a subset of genomes from mycocosm. Indeed, it's possible that the large dataset used here compensates for the above concerns, but without some attempt to evaluate these issues, it's difficult to have confidence in the results.

We very appreciate the positive reception of our manuscript. Following the reviewer’s comments we have investigated gene annotations in comparison with those of JGI Mycocosm, even though only 58 species were matching and only 19 of them were from the same strain. This dataset is not representative of the Sordariomycetes diversity (most species come from one clade), therefore will not reflect the results we obtained in this study. To note, the reason for not choosing JGI Mycocosm in the first place, was the poor representation of the insect-associated species, which we found key in this study. In general, we found that assembly lengths were nearly identical, number of genes was higher, and the repeat content was lower for the JGI Mycocosm dataset. When comparing different lifestyles (in particular pathogens vs. non-pathogens), we found the same differences for our and JGI Mycocosm annotations, with one exception being the repeat content. In the small subset (19 same-strain assemblies), our dataset showed the same level of repeats between the two lifestyles, whereas JGI Mycocosm showed lower repeat content for pathogens (but notably for all 58 species, the trend was same for our and JGI Mycocosm annotations). None of these observations are in conflict with our results where we find no or negative association of repeat content with pathogens. 

The figures are very information-dense. While I accept that this is somewhat of a necessity for presenting this type of study, if the authors could summarize the important information in easier-to-interpret plots, that could help improve readability.

We put a lot of effort into showing these complicated results in as approachable manner as possible. Given that other reviewers find them intuitive we decided to keep most of them as they are. To add more clarification, we added one supplementary figure showing distributions of genomic traits across lifestyles. Moreover, in Figure 5, a phylogenetic tree was added with position of selected clades, as well as a scatterplot showing distributions of mean values for genome size and number of genes for those clades. If the reviewer has any specific suggestions on what to improve and in which figure, we’re happy to consider it. 

Reviewer #2 (Recommendations for the authors):

I have no major comments on the analyses, which have already been extensively revised. My major criticism is the presentation of the background, which is very insufficient to understand the importance or relevance of the results presented fully.

Lines are not numbered, unfortunately, which will not help the reading of my review.

(1) The introduction could better present the background and hypotheses:

(a) After reading the introduction, I still didn't have a clear understanding of the specific 'genome features' the study focuses on. The introduction fails to clearly outline the current knowledge about the genetic basis of the pathogenic lifestyle: What is known, what remains unknown, what constitutes a correlation, and what has been demonstrated? This lack of clarity makes reading difficult.

We thank the reviewer for pointing this out. We have now included in the introduction a list of genomic traits we focus on. We also tried to be more precise about demonstrated pathogenic traits and other correlated traits in the introduction. 

(b) Page 3. « Various features of the genome have been implicated in the evolution of the pathogenic lifestyle. » The cited studies did not genuinely link genome features to lifestyle, so the authors can't use « implicated in » - correlation does not imply causation.

This sentence also somehow contradicts the one at the end of the paragraph: « we still have limited knowledge of which genomic features are specific to pathogenic lifestyle

We thank the reviewer for this comment. We added a phrase “correlated with or implicated in” and changed the last sentence of the paragraph into “Yet we still have limited knowledge of how important and frequent different genomic processes are in the evolution of pathogenicity across phylogenetically distinct groups of fungi and whether we can use genomic signatures left by some of these processes as predictors of pathogenic state.”.

(c) Page 3: « Fungal pathogen genomes, and in particular fungal plant pathogen genomes have been often linked to large sizes with expansions of TEs, and a unique presence of a compartmentalized genome with fast and slow evolving regions or chromosomes » Do the authors really need to say « often »? Do they really know how often?

We removed “often”.

(d) Such accessory genomic compartments were shown to facilitate the fast evolution of effectors (Dong, Raffaele, and Kamoun 2015) ». The cited paper doesn't « show » that genomic compartments facilitate the fast evolution of effectors. It's just an observation that there might be a correlation. It's an opinion piece, not a research manuscript.

We changed the sentence to “Such accessory genomic compartments could facilitate the fast evolution of effectors”.

(e) even though such architecture can facilitate pathogen evolution, it is currently recognized more as a side effect of a species evolutionary history rather than a pathogenicity related trait ». This sentence somehow contradicts the following one: « Such accessory genomic compartments were shown to facilitate the fast evolution of effectors".

Here we wanted to point out that even though accessory genome compartments and TE expansions can facilitate pathogen evolution the origin of such architecture is not linked to pathogenicity. We reformulated the sentence to “Even though such architecture can facilitate pathogen evolution, it is currently recognized that its origin is more likely a side effect of a species evolutionary history rather than being caused by pathogenicity”.

(f) As the number of genes is strongly correlated with fungal genome size (Stajich 2017), such expansions could be a major contributor to fungal genome size. » This sentence suggests that pathogens might have bigger genomes because they have more effectors. This is contradictory to the sentence right after « At the end of the spectrum are the endoparasites Microsporidia, which have among the smallest known fungal genomes ».

The authors state that pathogens have bigger genomes and then they take an example of a pathogen that has a minimal genome. I know it's probably because they lost genes following the transition to endoparasitism and not related to their capacity to cause disease. I just want to point out that their writing could be more precise. I invite authors to think of young scholars who are new to the field of fungal evolutionary genomics.

We thank the reviewer for prompting us to clarify the text. We rewrote this short extract as follows “Notably, not all pathogenic species experience genome or gene expansions, or show compartmentalized genome architecture. While gene family expansions are important for some pathogens, the contrary can be observed in others, such as Microsporidia. Due to transition to obligatory intracellular lifestyle these fungi show signatures of strong genome contractions and reduced gene repertoire (Katinka et al. 2001) without compromising their ability to induce disease in the host. This raises questions about universal genomic mechanisms of transition to pathogenic state.”

(g) I find it strange that the authors do not cite - and do not present the major results of two other studies that use the same type of approach and ask the same type of question in Sordariomycetes, although not focusing on pathogenicity:

Hensen et al.: https://pubmed.ncbi.nlm.nih.gov/37820761/

Shen et al.: https://pubmed.ncbi.nlm.nih.gov/33148650/

We thank the reviewer for pointing out this omission. We now added more information in the introduction to highlight the importance of the phylogenetic context in studying genome evolution as demonstrated by these studies. The following part was added to introduction:  “Other phylogenomic studies investigating a wide range of Ascomycete species, while not explicitly focusing on the neutral evolution hypothesis, have found strong phylogenetic signals in genome evolution, reflected in distinct genome characteristics (e.g., genome size, gene number, intron number, repeat content) across lineages or families (Shen et al. 2020; Hensen et al. 2023). Variation in genome size has been shown to correlate with the activity of the repeat-induced point mutation (RIP) mechanism (Hensen et al. 2023; Badet and Croll 2025), by which repeated DNA is targeted and mutated. RIP can potentially lead to a slower rate of emergence of new genes via duplication (Galagan et al. 2003), and hinder TE proliferation limiting genome size expansion (Badet and Croll 2025). Variation in genome dynamics across lineages has also been suggested to result from environmental context and lifestyle strategies (Shen et al. 2020), with Saccharomycotina yeast fungi showing reductive genome evolution and Pezizomycotina filamentous fungi exhibiting frequent gene family expansions. Given the strong impact of phylogenetic membership,  demographic history (Ne) and host-specific adaptations of pathogens on their genomes, we reasoned that further examination of genomic sequences in groups of species with various lifestyles can generate predictions regarding the architecture of pathogenic genomes.”

(h) Genome defense mechanisms against repeated elements, such as RIP, are not mentioned while they could have a major impact on genome size (Hensen et al cited above; Badet and Croll https://www.biorxiv.org/content/10.1101/2025.01.10.632494v1.full).

This citation is added in the text above.

(i) Should the reader assume that the genome features to be examined are those mentioned in the first paragraph or those in the penultimate one?

In the last paragraph of the introduction we included the complete list of investigated genomic traits.

(j) The insect-associated lifestyle is mentioned only in the research questions on page 4, but not earlier in the introduction. Why should we care about insect-associated fungi?

We apologize for this omission. We added a sentence explaining how neutral evolution hypotheses can explain patterns of genome evolution in endoparasites and species with specialized vectors (traits present in insect-associated species) and added a sentence in the last paragraph that this is the reason why we have selected this trait for analysis.  

(2) Why use concatenation to infer phylogeny?

(a) Kapli et al. https://pubmed.ncbi.nlm.nih.gov/32424311/ « Analyses of both simulated and empirical data suggest that full likelihood methods are superior to the approximate coalescent methods and to concatenation »

(b) It also seems that a homogeneous model was used, and not a partitioned model, while the latter are more powerful. Why?

We thank the reviewer for the comment. When we were reconstructing the phylogenetic tree  we were not aware of the publication and we followed common practices from literature for phylogenetic tree reconstruction even though currently they are not regarded as most optimal. In fact, in the first round of submission, we have included both concatenation as well as a multispecies coalescent method based on 1000 busco sequences and a concatenation method with different partitions for 250 busco sequences. All three methods produced similar topologies. Since the results were concordant, we chose to omit these analyses from the manuscript to streamline the presentation and focus on the most important results.

(3) Other comments:

Is there a table listing lifestyles?

Yes, lifestyles (pathogenicity and insect-association) are listed in Supplementary Table S1. 

(4) Summary:

(a) seemingly similar pathogens »: meaning unclear; on what basis are they similar? why « seemingly »?

We removed “seemingly” from the sentence.

(b) Page 4: what's the difference between genome feature and genome trait?

There is no difference. We apologize for the confusion. We changed “feature” to “trait” whenever it refers to the specific 13 genomic traits analyzed in this study.

(c) Page 22: Braker, not Breaker

corrected

What do the authors mean when they write that genes were predicted with Augustus and Braker? Do they mean that the two sets of gene models were combined? Gene counts are based on Augustus (P24): why not Braker?

We only meant here that gene annotation was performed using Braker pipeline, which uses a particular version of Augustus. We corrected the sentence.

(d) Figure 2B and 2C:

'Undetermined sign' or 'Positive/Negative' would be better than « YES » or it's just impossible to understand the figure without reading the legend.

We changed “YES” to “UNDETERMINED SIGN” as suggested by the reviewer.

This sentence captures the essence of the transactional model by showing how communication is a collaborative, continuous process where meaning is co-created rather than simply transmitted

this sentence underscores how personal and cultural differences shape interpretation, showing that effective communication depends on understanding others’ perspectives and shared experiences

This highlights how external or internal distractions can disrupt understanding, reminding readers that effective communication depends not only on clear messages but also on managing barriers that alter meaning

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Discussion

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eLife Assessment

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