Reviewer #3 (Public review):

Summary:

In this study, authors Simone Rencken and co-authors present and investigate the genome of the common cuttlefish Sepia officinalis.

Strengths:

The authors explain in a detailed yet concise manner the main steps for a genome assembly, with very robust methods for validation, and according to current best practices. In addition to the chromosomal assembly, the authors confirmed the presence of 47 chromosomes using Hi-C data and multiple species synteny. They also generated a comprehensive gene annotation, with assessments of gene completeness, providing a useful resource for the community of researchers interested in cuttlefish biology and comparative genomics.

Author response:

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

Public Reviews:

Reviewer 1 (Public review):

Summary:

This manuscript presents a high-quality, chromosome-level genome assembly of the European cuttlefish (Sepia officinalis), a representative species of the cephalopod lineage. Using state-of-the-art sequencing and scaffolding technologies -including PacBio HiFi long reads and Hi-C chromatin conformation capture - the authors deliver a genome assembly with exceptional contiguity and completeness, as evidenced by high BUSCO scores. This genome resource fills a significant gap in cephalopod genomics and offers a valuable foundation for studies in neurobiology, behavior, and evolutionary biology. However, there are several major aspects that need to be strengthened.

Major Revisions Recommended:

(1) Single-individual genome limitation

The genome assembly is based on a single individual, which appears to be male. While this approach is common in genome projects, it does not capture the full genetic diversity of the species. As S. officinalis exhibits a wide geographical range and possible population structure, future efforts (or discussion in this manuscript) should consider re-sequencing multiple individuals - of both sexes and from diverse geographic origins - to characterize population-level variation, sex-linked features, and structural polymorphisms.

We thank the reviewer for this summary and the important point raised. While sequencing additional individuals, unfortunately, lies outside the scope of our study, we used the published data from the DToL assembly (from a male individual from a different geographical origin) to begin to investigate their differences.

First, we attempted to create a mixed assembly from both datasets, as also suggested by Reviewer 2, to increase data coverage and genetic information. Even though the heterozygosity estimate is quite low (ca. 1%), the mixed assembly produced severely inflated and fragmented results, yielding an assembly ca. 3× larger than expected, with the top 46 contigs covering only ~5% of the total length - a sign of over duplication and failed haplotype collapse.

This result is not surprising when considering the assembly algorithms: most programs, including hifiasm used in this study, assume a single diploid individual (or a trio assembly including data from both parents), so using multiple individuals breaks this assumption. Assembly pipelines infer homozygous/heterozygous coverage cutoffs from the k-mer histogram. Mixing individuals raises apparent heterozygosity far above true diploid levels, turning the expected bimodal k-mer profile into a complex multimodal distribution. This misleads the phasing and purging steps in the assembly pipeline, causing over-expansion and fragmentation of the assembly.

Second, we created separate assemblies from the raw data sets of MPIBR and DToL using the exact same pipeline and parameters to avoid the technical problem described above. These assemblies are directly comparable, and after aligning them, it is possible to build a pangenome graph that we believe would help to address the points raised by the reviewer. Pangenome graphs can represent cross-individual variation more accurately and improve read alignment in regions of high genomic variation, which can aid population-level analyses [1]. We agree on the importance of this work, yet collecting data from more individuals and the construction and analysis of a pangenome graph lies beyond the scope of this manuscript and should be part of future efforts by the cephalopod genomics field.

(2) Limited experimental validation of chromosomal inferences

The study reports chromosome-scale scaffolding using Hi-C data and proposes a revised karyotype for S. officinalis. However, these inferences would be significantly strengthened by orthogonal validation methods. In particular, fluorescence in situ hybridization (FISH) or karyotyping from cytogenetic preparations would provide direct confirmation of chromosome number and structural arrangements. The reliance solely on Hi-C contact maps for inferring chromosomal organization should be acknowledged as a limitation or supplemented with such validations.

We appreciate the reviewer’s point regarding the value of orthogonal validation methods to support the chromosome-scale scaffolding and proposed karyotype. We acknowledge that relying solely on Hi-C contact maps to infer chromosome number and structure presents limitations, as also becomes apparent in our detailed analysis of both S. officinalis genome assemblies (in Figure 2 and Supplementary Figure 3 of the revised manuscript). We attempted to complement these analyses with cytogenetic approaches. Unfortunately, the availability of suitable mitotic tissue was limited. Moreover, our karyotyping trials proved challenging: resolving the ≥92 (2n) chromosomes in situ was not feasible due to their high number and the small size of the nuclei (approximately 5 µm in diameter on average).

We now highlight this point as an important direction for future work in our discussion (line 456-466):

“Additional methods such as cytogenetic karyotyping or optical mapping such as BioNano [141] (imaging of fluorescently tagged, linearized DNA) could be used to validate chromosome numbers. However, whereas karyotypes of octopuses have been consistent throughout the literature (1n=30) [142,143], those measured in decapods vary greatly. For example, 1n=46 chromosomes have been reported for two species of cuttlefish (A. esculentum and A. lycidas) and three loliginid squids [85]; 1n=36 has been reported for A. Arabica [86] and 1n=24 in A. pharaonis [87]. In S. officinalis, a karyotype of 1n=52 is reported for testis samples [88]. Combining cytogenetic preparations with fluorescent labeling of centromeric or telomeric sequences, as demonstrated in the octopus A. aerolatus [143] could help resolve these issues. Establishing a routine staining protocol would enable comprehensive tests at the species- and population-level.”

(3) Shallow discussion of chromosomal evolution

The manuscript briefly mentions chromosomal number differences among cephalopods but does not explore their evolutionary or functional implications. A more thorough comparative analysis - linking chromosomal rearrangements (e.g., fusions, fissions) with ecological adaptation, life history, or neural complexity - would greatly enhance the impact of the findings. Referencing chromosomal dynamics in related taxa and possible links to behavioral innovations would contextualize these results more effectively.

We agree with the reviewer that this is a fascinating topic of research that demands further attention and have extended our discussion, which now reads (line 476-501):

“In addition to studying chromosomal topology in phylogenetic reconstructions, some of the most interesting aspects of these rearrangements relate to changes of and innovation in regulatory elements that underlie phenotypic diversity. In coleoid cephalopods, it is thought that an ancient large-scale genome rearrangement was combined with lineage-specific changes and repeat expansions [48–50]. This restructuring gave rise to hundreds of tightly linked, evolutionarily unique microsyntenies, corresponding to distinct topological compartments with specialized regulatory architectures that contribute to complex, tissue-specific expression patterns in the nervous system and elsewhere [43]. Extending this, chromosomal conformation analyses in E. scolopes revealed that co-regulated eye and light-organ genes cluster at topologically associating domain (TAD) boundaries, and that an evolutionarily recent rearrangement at the dachshund (DAC) locus may have been instrumental in the emergence of the symbiotic light organ in Euprymna - directly linking specific chromosomal topology to morphological innovation [44].

To understand the broader functional impact of these changes across coleoids, a recent study investigating Micro-C, RNA-seq, and ATAC-seq data from multiple species revealed broadly conserved chromatin domains, but also many lineage-specific chromatin loops that form novel regulatory signatures and impact expression profiles across species and tissues [149].

Despite the observed small-scale regulatory changes, the chromosomes of decapods are considered to be more closely related to the ancestral coleoid karyotype than those of octopods. The derived octopod karyotype becomes apparent when comparing it to the genome of the vampire squid, an early-branching octopodiform (sister to all octopods) which retained features of the decapod, ancestral karyotype [150]. Taken together, the conserved karyotype of decapods accommodates fine-scale regulatory diversity that might underlie morphological diversity among species, which suggests that many regulatory innovations are still being evolutionarily explored through rearrangements within the existing chromosomes.”

(4) Underdeveloped gene family and pathway analysis

While the authors identify expansions in gene families such as protocadherins and C2H2 zinc finger transcription factors, the functional significance of these expansions remains speculative. The manuscript would benefit from:

(a) Functional enrichment analyses (e.g., GO, KEGG) targeting these gene families.

(b) Expression profiling across tissues or developmental stages to infer regulatory roles.

(c) Comparison with expression or expansion patterns in other cephalopods with known behavioral complexity (e.g., Octopus bimaculoides, Euprymna scolopes).

(d) Potential integration of transcriptomic or epigenomic data to support regulatory hypotheses.

We thank the reviewer for these constructive suggestions and have substantially expanded the functional characterization of expanded gene families in the revised manuscript.

To address points a) + b), we performed GO enrichment analyses for all expanded gene families (orthogroups), both for the largest gene families and the most significantly expanded families identified from our CAFE5 analysis. Further, we cross-referenced all S. officinalis members of each expanded orthogroup against differentially expressed genes in our bulk RNA-seq data from multiple tissues (initially collected to improve the gene modeling), allowing us to infer tissue-specific expression patterns for the expanded families.

To address point (c), the species-resolved copy-number profiles from our orthogroup analysis directly situate the S. officinalis expansions within the broader coleoid context, including O. bimaculoides, O. vulgaris, E. scolopes, and D. pealeii, enabling direct comparison of expansion scale and lineage specificity across species with varying degrees of behavioural complexity. We note that the C2H2 zinc finger and protocadherin expansions show distinct phylogenetic profiles consistent with independent radiations in octopods and decapodiforms, in agreement with recent studies.

Regarding point (d), no epigenomic data for S. officinalis was publicly available at the time of writing, thus we focused on the transcriptomic data from this study, as described above.

We describe this analysis in two additional results paragraphs to the manuscript, one modified (Figure 4) and two new figures (Figure 5 and Supplementary Figure 7), which are reproduced (lines 294-400):

“Analysis of expanded gene families

We sought to investigate the S. officinalis gene annotation and place it in the context of gene repertoires from other cephalopod or molluscan species. First, we collected available genome annotations from 12 other molluscan species (Table 2) and clustered them using OrthoFinder v.3.1.0 [122], resulting in 23,658 orthogroups, hereafter named gene families.

First, we investigated 36 of the gene families that contain more than 100 genes in any of the species, with 17 of these families containing at least one gene of S. officinalis, that reflect large-scale gene family expansions (Figure 4E). We used the InterProScan and eggNOG-mapper annotations to infer functional roles of these genes, selecting the most common gene annotation as the name of the gene family.

The zinc finger C2H2-type transcription factors (TFs) were grouped into three of the large gene families, with the largest family (OG0000000) only present in decapod cephalopods. This likely reflects the largely independent expansions in the octopod and decapod lineages that date back to a burst of transposon activity ca. 25 million years ago [46,48,49]. The largest expansion across mollusks occurs in the cadherin-like family (OG0000001): 310 in S. officinalis, 283 in D. pealeii, 209 in A. lycidas, 102 in O. vulgaris, 55 in O. bimaculoides, with low but non-zero counts in bivalves (C. virginica, M. gigas). This profile is consistent with the protocadherin expansion first described in O. bimaculoides [46] and subsequently shown to be present across cephalopods [48,49,123].

HPGDS (OG0000005, hematopoietic prostaglandin D synthase) is a glutathione-S-transferase family member that catalyzes the conversion of prostaglandins, which have well-described roles in immune responses in vertebrates and insects [124,125]. This family shows a broad expansion in decapods, with a lesser expansion in octopods. Additionally, members of the glutathione-S-transferase families have been co-opted as S-crystallins, structural proteins found in the lens of cephalopods that may, or may not, retain enzymatic functions [126,127].

Two large families are mostly lineage-restricted. The RING-type zinc finger family (OG0000058) has 103 copies in S. officinalis and 26 in A. lycidas but is absent in all other species except for E. scolopes. Conversely, OG0000002 (unknown function) has 479 copies in E. scolopes and only a few copies in the other species. This interesting Sepiolid-specific expansion warrants further characterization.

We estimated gene family evolution rates using CAFE5 [128] for all families with less than 100 copies in any species (this excludes the families described above, as very large copy-number differences between species preclude likelihood calculations under the applied birth-death model). After comparing different model parameters, we chose a gamma model with three rate categories, allowing for evolutionary rate variation among gene families. Out of the 12,895 gene families analyzed, 1,813 showed a significant (p < 0.05) expansion or contraction in at least one of the species. We focused our analysis on the 30 most significantly expanded families; among them were several retrotransposon-associated domains that have expanded specifically in S. officinalis five families carrying Retrovirus-related Pol polyprotein domains, two Reverse transcriptase domain families, and four Ribonuclease H-like families (Supplementary Figure 7A). There was no coordinate-based overlap of the coding sequences with annotated TEs from the RepeatMasker output (Methods).

In addition to the three large gene families of C2H2 zinc finger expansions, 45 gene families containing this TF type showed a significant change in the CAFE5 analysis. Notably, eight of the significant gene families, as well as four of the largest gene families, were annotated as CCHC-type zinc fingers, which contain a “zinc knuckle” motif that is characteristic of retroviral nucleocapsid proteins [129] and is functionally integrated in the genomes of several species, including humans [130].

Some gene families without any relationship to retrotransposons were also expanded. For example, the UGT2A1-related family is a UDP-glucuronosyltransferase, a class of enzymes central to phase II detoxification and conjugation of metabolites, reported in other mollusks in the context of environmental chemical tolerance [131], and in insects in the context of pigmentation [132]. We also detected a family of homeodomain-like proteins, representing an expansion of this important TF family.

Tissue-specific expression of expanded gene families

To place the identified gene families in a functional context, we profiled their expression in the bulk RNA-seq data (taken from multiple tissues of S. officinalis) used originally for gene modeling (Figure 5A). Principal component analysis (PCA) revealed the largest axis of variation in gene expression to separate brain tissues from peripheral tissues, with skin being the most transcriptomically distinct (Figure 5A), consistent with the high number of tissue-specific differentially expressed (DE) genes identified in non-neural tissues (Figure 5B). We identified the genes belonging to expanded families that were differentially expressed across tissues and enriched gene ontology [133,134] (GO) terms for them to gain additional insight. The large families excluded from CAFE5 modelling and the significantly expanded families identified by CAFE5 were analyzed separately.

Eleven of the largest gene families were expressed in our data (Figure 5C) and five had enriched GO terms (Figure 5D,E). Among them, the cadherin family showed brain-restricted expression and GO terms related to cell–cell adhesion and calcium binding, consistent with their role in neuronal connectivity and circuit formation [46,135]. Two C2H2 zinc finger gene families were expressed in the optic and vertical/subvertical lobes of the brain and in the skin, with GO terms related to DNA-binding, transcriptional regulation or development. The RING-type zinc finger family was expressed specifically in the skin, with GO terms including zinc binding and ubiquitin protein ligase activity, the canonical function of RING-domain E3 ligases [136]. Genes of the HPGDS/S-crystallin family were expressed in the brain (basal and optic lobes and posterior subesophageal mass) and skin, with GO terms related to glutathione metabolism, matching their described enzymatic function. We did not find expression in the retina, which is expected given that S-crystallins are expressed in lentigenic cells of the eye [42,137] and these cells were not included during sampling.

Among the 30 most significantly expanded families examined (out of 1,813 total), expression was widespread (20/30) and tissue-specific differential expression was common (17/30), suggesting that a substantial proportion of expanded paralogs represent functional coding sequences with specialized spatial deployment (Supplementary Figure 7B). Ten of the retrotransposon-associated families were differentially expressed in the brain (optic and vertical/subvertical lobes) and skin, arguing against these loci being inactive repeat fragments and supporting their inclusion as transcribed gene models. Two significantly expanded families showed both differential expression and enriched GO terms (Supplementary Figure 7C). The first was the UGT2A1-related family, which had the largest number of differentially expressed genes overall, with expression concentrated in the skin, retina and posterior subesophageal mass of the brain. Enriched GO terms matched the described enzymatic function for this family, namely UDP-glycosyltransferase activity. The second gene family was the homeodomain-like family with enrichment for DNA binding terms consistent with their role as transcription factors, and was preferentially expressed in the vertical and subvertical brain lobes with weaker expression in other areas.

Collectively, many differentially expressed genes from expanded families were restricted to specific tissues or brain subregions (Figure 5F and Supplementary Figure 7D), indicating that paralogs within an expanded family have adopted distinct spatial expression domains and possibly, specialized functions.”

Reviewer 2 (Public review):

Summary:

This paper concerns an interesting organism, Sepia officinalis. However, in the opinion of this reviewer, the paper reads somewhat like a genome report. The authors have used 23x PacBio HiFi in conjunction with relatively low coverage (11x) Hi-C to scaffold the genome into a karyotype of 47 chromosomes. They have used a combination of short and long read RNA seq to annotate the genome in what looks like a very good annotation. The paper offers basic analyses of the Busco evaluation, some descriptive analyses of gene family and repeat content, and a bit more focused analysis on synteny among sequenced squids. Generally, the data will be useful.

Strengths:

This is a high-quality annotation, and the data ultimately will be useful to other researchers. I appreciate trying to understand what's happening between assemblies of S. officinalis.

Weaknesses:

I don't believe the data at hand makes a strong case for the argument of 47 chromosomes. This is my biggest sticking point with the paper, and it is for a few reasons:

(1) The authors point to assembly differences between the DToL assembly and the one presented in the manuscript and seem to claim that DToL is incorrect. However, the DToL assembly (xcSepOffi3.1) is based on much deeper HiFi and HiC coverage than the one at hand (51x and 80+x respectively). There are many things to try here, including:

(a) Downloading the DToL data and reassembling using a common pipeline.

(b) Downsampling the DToL data to similar coverage as what the authors have achieved.

(c) Combining your data and that of DToL for even deeper coverage (heterozygosity is low enough that I don't imagine this impeding things too badly).

We thank the reviewer for these helpful suggestions and want to clarify that we did not seek to point out errors in the DToL assembly, but rather to investigate the unexpected discrepancies between the two assemblies. It is correct that the DToL data has a much higher coverage than our data. We followed the individual suggestions and incorporated them into the revised manuscript. We reproduce the relevant sections below, and provide additional information:

(a) Downloading the DToL data and reassembling using a common pipeline.

We downloaded the DToL data and reassembled it using a common pipeline, yielding the results listed in Author response table 1. The DToL assembly is more contiguous, which is mainly due to its higher HiFi coverage. It also receives slightly better BUSCO scores (computed using odb12 as recommended by Reviewer 3).

Author response table 1.

Full statistics of S. officinalis assemblies from two independent datasets, assembled using a common pipeline.

The updated manuscript now reads (lines 146-159):

“A chromosome-scale assembly for Sepia officinalis was released recently by the Wellcome Sanger Institute’s Darwin Tree of Life project [75] (DToL, GCA_964300435.1). That genome was assembled from a male individual using high coverage PacBio Sequel II (~51x) and Arima2 Hi-C (~80x) data, with a final assembly size of 5.8 Gb. The the haploid chromosome number was estimated to be 49. To compare both S. officinalis datasets directly, we downloaded the DToL data and created two new assemblies using the pipeline described above (hifiasm using PacBio HiFi and Hi-C data). The resulting assemblies were overall very similar, with the DToL assembly having a slightly higher contiguity (N50 length, see Table 1) and BUSCO completeness (Supplementary Figure 2A,B) due to their higher sequencing coverage.”

To further compare the two datasets, we added a new Figure 2 to the revised manuscript and the following paragraph to the results (lines 160-169):

“After scaffolding with YAHS, both datasets reached the previously identified chromosome numbers (1n=47 for MPIBR and 1n=49 for DToL, Figure 2A,B). To further investigate this surprising discrepancy, we aligned both assemblies using Winnowmap [89] to locate the differences between them (Figure 2C). We observed four “breakpoints” (BP) of chromosome scaffolds: one in the MPIBR assembly compared to DToL (BP1: DToL_5 = MPIBR_40+44) and three in the DToL assembly compared to MPIBR (BP2: DToL_31+40 = MPIBR_2, BP3: DToL_41+46 = MPIBR_6, BP4: DToL_44+45 = MPIBR_7). We also aligned the assemblies to the chromosome-scale genome of another cuttlefish Acanthosepion esculentum (1n=46, GCA_964036315.1). In this alignment, all four breakpoints were collinear with single A. esculentum chromosomes (Figure 2D).”

(b) Downsampling the DToL data to similar coverage as what the authors have achieved.

Instead of downsampling the DToL data, we decided to analyze the Hi-C and HiFi data for both assemblies, focusing on the four “breakpoints” between the assemblies and the A. esculentum genome that we described above. First, we performed a QC analysis of the Hi-C reads using pairtools [2], the result is visualized in Author response image 1. The percentage of valid Hi-C read pairs, i.e., cis pairs with insert distances of more than 1 kb and trans pairs, following the Dovetail genomics QC manual (https://dovetail-analysis.readthedocs.io/en/latest/whole_genome/qc.html). When Hi-C pairs were aligned to the primary contigs from hifiasm (as is used for scaffolding with YAHS), the DToL HiC data contains fewer valid read pairs (11.4%) than the MPIBR data (43.1%), possibly due to using a different tissue (eye vs. optic lobe) and HiC kit (Arima 2 vs. Dovetail OmniC) for the library preparation. Nonetheless, due to the much higher overall coverage, the amount of valid read pairs is still 2.35x higher for DToL (144,014,368 pairs) than for MPIBR (61,318,955 pairs). The higher trans fraction (i.e. HiC pairs across contigs) is dependent on the length of the primary contigs, so the higher trans fraction for the MPIBR data can be explained by the lower contiguity of its primary contigs. It is conceivable that for both assemblies, the low numbers of valid read pairs introduce a technical fragmentation of certain chromosomes, as indicated by the identified breakpoints (Figure 2).

Author response image 1.

Analysis of Hi-C read pairs from both S. officinalis assemblies. Hi-C reads were aligned to the primary contigs from hifiasm (as is used for scaffolding with YAHS) and analyzed using pairtools. Note the higher fraction of long-range contacts (at least 1 kb cis pairs or trans pairs) in the MPIBR data (top) compared to DToL (bottom). Due to overall higher coverage, the absolute number of read pairs is higher for DToL than for MPIBR data.

Second, we performed a detailed analysis of read coverage along the breakpoint junctions of the discrepant chromosomes/scaffolds between both assemblies. We included a description of the results and a new Supplementary Figure 3 in the manuscript, (lines 171-207):

“To better understand the potential cause of these divergent chromosome numbers, we analyzed the Hi-C and HiFi coverage in the breakpoint regions (Supplementary Figure 3A). First, we aligned the Hi-Fi reads to the scaffolds and extracted all alignments along the 200 kb terminal scaffold windows to find any notable drops in coverage, or reads spanning any of the scaffold junctions. We detected no spanning reads. This is not surprising given that no contigs were assembled at these sites, resulting in the observed scaffold junctions. More interestingly, we noted a ~5-fold decrease in HiFi coverage along the DToL scaffold_40 (part of BP2) relative to its flanking regions, indicating a highly repetitive, low-mappability region at this boundary.

Next, we realigned the Hi-C data to the scaffolded assemblies using bwa-mem2 [91] and extracted all trans HiC pairs (between-scaffold contacts) using pairtools [92]. We normalized trans HiC contacts to the scaffold length and compared contact rates between breakpoint scaffolds to the baseline contact rate (computed from pairs of scaffolds with a clear 1-to-1 match between assemblies), and the contact rate within scaffolds (intra-scaffold pairs) (Supplementary Figure 3B,C). The contact rates within breakpoints were consistently lower than within scaffolds, likely falling below the threshold to be merged during assembly. However, the contact rates at three of four breakpoints (BP1, BP3, BP4) were significantly elevated above the genome-wide background distribution (empirical p = 0.010, 0.005, 0.005 respectively), suggesting that they may represent intra-chromosomal contacts disrupted by a misassembly. Notably, BP2 was not significant (empirical p = 0.170), likely due to the low coverage and mappability around the DToL scaffold_40 boundary. Considered jointly, the three DToL breakpoint scaffold pairs showed significantly higher trans contact rates than the background (Wilcoxon rank-sum, one-tailed, U = 1771, p = 0.004).

Lastly, we analyzed the repeat landscape around the 200 kb scaffold ends using RepeatMasker [93] and the custom repeat library that we had generated for Sepia officinalis (described further below). Compared to control scaffolds of the same assembly, we observed consistently elevated repeat content at the breakpoint junctions (mean 71.5% vs 67.6% masked bases), with an enrichment of unclassified repeats (32.1% vs 30.0%), which could explain a repeat-driven assembly fragmentation or scaffolding failure. The BP2 DToL scaffold_40 junction window was 99.99% masked (99.2% unclassified repeats), providing a likely mechanistic explanation for both the HiFi coverage drop and the absence of a significant trans Hi-C signal at this breakpoint. Taken together, these analyses suggest that the different chromosome numbers across the two S. officinalis assemblies are due to technical reasons, caused by repeat-rich scaffold boundaries that impair HiFi and Hi-C read alignment and in turn, correct assembly in these regions.”

(c) Combining your data and that of DToL for even deeper coverage (heterozygosity is low enough that I don't imagine this impeding things too badly).

When combining the data to achieve a higher coverage, we ran into the assembly fragmentation issues detailed above in response 1) to Reviewer 1.

(2) Looking at Figure 1, there appears to be a misjoin at chromosome 42. Looking carefully at Figure S1, that misjoin does not appear on any of the panels - this is confusing. Given the size of that chromosome and the authors' chromosome numbering, I'm guessing this is a manual merge (as it's larger than most of the chromosomes numerically close (40, 41, 43, etc). Further, staring closely at Figure 1, there appear to be cross-scaffold contacts between 42 and 43 and 42 and 44. Secondarily there are contacts between 43 and 44. This bit of the assembly seems potentially problematic.

This is a great observation, indeed the HiC maps differ between Figure 1 and Figure S1. Figure 1 is the result of scaffolding with YAHS and manual curation, whereas Figure S1 was scaffolded using HapHiC. We updated the figure legend to clarify this important difference. HapHiC produces very clean contact maps without the need for manual curation, but when analyzed at a higher resolution, the tool broke many contigs and ultimately compromised the assembly quality, possibly due to our comparatively low HiC coverage. Thus, we preferred to use YAHS and manual curation, which is perhaps inherently error-prone, as becomes apparent in the regions of the assembly that are pointed out by the reviewer.

Reviewer 3 (Public review):

Summary:

In this study, authors Simone Rencken and co-authors present and investigate the genome of the common cuttlefish Sepia officinalis.

Strengths:

The authors explain in a detailed yet concise manner the main steps for a genome assembly, with very robust methods for validation, and according to current best practices. In addition to the chromosomal assembly, the authors confirmed the presence of 47 chromosomes using Hi-C data and multiple species synteny. They also generated a comprehensive gene annotation, with assessments of gene completeness, providing a useful resource for the community of researchers interested in cuttlefish biology and comparative genomics.

Weaknesses:

While the study touches upon the subjects of gene content, TE activity, or species-level comparisons, the study does not provide in-depth investigations of these.

We thank the reviewer for their positive assessment of our manuscript. We acknowledge the descriptive nature and limitations of our previous analyses of gene content, TE distribution, and species comparisons. Our focus for the initial submission was to provide a high-quality assembly that could serve as a resource for anyone interested in Sepia officinalis or related species. However, we agree that greater insight into genome content is valuable as well. In the revised manuscript, we included a more detailed analysis of expanded gene families and GO enrichment analysis of our bulkRNAseq data, which we summarized in response 4) to reviewer 1.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Minor Revisions Recommended:

(1) Figure and legend clarity

Several figures lack sufficient annotation. All figures, including supplementary ones, should include:

(a) Clear axis labels.

(b) Descriptions of statistical measures (n values, error bars, statistical tests).

(c) Legends that allow the figure to be understood independently of the main text.

We updated the figures accordingly.

(2) Terminology and formatting

(a) Consistency in gene and species nomenclature should be maintained throughout (e.g., italicizing gene names and Latin binomials).

(b) Ensure that abbreviations (e.g., Hi-C, BUSCO, FISH) are defined upon first use.

We updated the nomenclature throughout the text and checked the definition of abbreviations used in the text. Further, we updated the names of several cuttlefish species according to the recent revision of genera, e.g. Sepia esculenta was changed to Acanthosepion esculentum [3].

(3) Literature coverage

The references primarily focus on earlier studies from 2010-2020. It would strengthen the context to include recent high-impact studies on cephalopod genomics and chromosomal biology published in the last 3 years (e.g., 2022-2024).

We apologize for this oversight and have extended the manuscript to discuss more of these recent studies.

(4) Clarify methods

While the methods section is generally detailed, some critical aspects are underspecified:

(a) Parameters used in genome annotation tools (e.g., BRAKER, RepeatMasker).

We thank the reviewer for bringing our attention to this shortcoming, and have added the missing parameters to the methods section. Additionally, the full code is available at https://gitlab.mpcdf.mpg.de/mpibr/laur/cuttlefishomics/soffgenome

(b) Criteria for ortholog clustering and gene family expansion analysis.

The details have been added to the methods section, which now reads (lines 828-853):

“Orthogroups were inferred across 13 molluscan species (Table 2), including S. officinalis, using OrthoFinder v3.1.0 [122] with default parameters. The input proteomes included the longest protein isoform per gene for each species. The rooted species tree from OrthoFinder [182,184] was converted to an ultrametric tree using the R package ape [183] v5.8.1.

Gene families were filtered by removing orthogroups present in only a single species, and by separating orthogroups containing 100 or more gene copies in any species, as extreme copy-number differences in gene families prevent likelihood calculation under the applied birth-death model.

Gene family evolution rates were estimated using CAFE5 [128] v5.1.1 on the filtered orthogroups, using the ultrametric species tree as input. Four models were evaluated: the base model (single global lambda), and Gamma models with k = 2, 3, and 4 rate categories, which allow evolutionary rate variation among gene families. The Gamma k = 3 model was selected based on the best (lowest) final log-likelihood score. All subsequent statistical inferences were performed under this model.

For families showing statistically significant expansion or contraction (p < 0.05 after Bonferroni correction), branch-specific copy-number changes were extracted from the CAFE5 output. Families were categorized as S. officinalis-specific, coleoid-specific, or broad expansions based on the distribution of significant changes across the phylogeny.

To assess whether expanded gene families in S. officinalis contained genes derived from or embedded within repetitive elements, a coordinate-based overlap analysis was performed. For each gene in an expanded orthogroup, the overlap between its coding sequence (CDS) coordinates and RepeatMasker annotations was computed using bedtools intersect v2.30 [185]. To avoid double-counting when multiple repeat annotations overlapped the same coding bases, overlapping repeat intervals were merged per gene prior to summing covered bases, and the overlap fraction was computed as merged covered bases divided by total CDS length.”

(c) Thresholds or cutoffs for synteny or duplication detection.

We included the details in the updated methods (lines 755-781):

“Synteny analyses between all chromosomes of the compared species were performed using the R package GENESPACE v.1.2.3 [175] with default parameters, described briefly below. Protein sequence similarity was first estimated using DIAMOND2 [109] in fast mode, and orthogroups and pairwise orthologues were inferred using OrthoFinder v2.5 [176] with hierarchical orthogroups (HOGs) enabled. Prior to synteny inference, tandem arrays were condensed to their most central representative gene, and gene rank order was recalculated on these array-representative genes to reduce confounding effects of tandem duplication on collinearity detection.

Syntenic blocks were identified pairwise between all genome combinations using MCScanX [177], constrained to DIAMOND hits where both query and target genes belonged to the same orthogroup (onlyOgAnchors = TRUE). Initial anchor hits were clustered into large syntenic regions using a density-based spatial clustering approach (dbscan [178]), with a minimum block size of five anchor genes (blkSize = 5) and a maximum of five intervening non-anchor genes permitted within a block (nGaps = 5). Anchor clustering used a search radius of 25 gene-rank positions (blkRadius = 25). All hits falling within a syntenic buffer of 100 gene-rank positions around confirmed block anchors (synBuff = 100) were retained as syntenic. No secondary syntenic hits were included (nSecondaryHits = 0). Syntenic orthogroups were integrated across all pairwise comparisons and collapsed into a pan-genome annotation anchored to. S. officinalis was used as the reference genome.

Syntenic relationships were visualized as riparian plots and pairwise dotplots using the built-in plotting functions of GENESPACE v1.2.3. Riparian plots were constructed using physical chromosomal coordinates (useOrder = FALSE) with S. officinalis as the reference, displaying all three genomes. A second riparian plot was generated highlighting a region of interest. Pairwise dotplots were produced species for the S. officinalis–D. pealeii and S. officinalis–E. scolopes genome comparisons, displaying only synteny-validated hits (type = "syntenic") with a minimum synteny score of 10 (minScore = 10) and a minimum of 10 genes per chromosome pair required for display (minGenes2plot = 10).”

Reviewer #2 (Recommendations for the authors):

Line 153 should be supplemental Figure 3B.

The text was referring to the correct Figure 2B (three species synteny comparison). It is now updated to Figure 3B in the revised manuscript.

Reviewer #3 (Recommendations for the authors):

(1) L37: Perhaps add a comparison with other species (mammals, Drosophila, etc.) to put this number in context.

We agree with this recommendation and added numbers for Drosophila and mouse to the text (lines 40-45):

“Coleoid cephalopods (octopus, squid, cuttlefish) are a highly derived group of mollusks, characterized by the largest nervous systems among all invertebrates (ca. 500 million neurons in an adult octopus of which 200 million are in the central brain [1,2], compared to ca. 140,000 in the fruit fly [3] or 70 million in the mouse [4]) and specializations with a great historical importance for neuroscience (e.g., “giant axons” [5] and “giant synapses” [6–8]).”

(2) L51, 279: "Octopodiformes" is a superorder, not a genus or a species name. It should not go in italics.

We updated this throughout the text.

(3) L53: "even smaller" seems odd here, because the argument of the sentence is to stress the large genome size of Octopodiformes. Perhaps start the sentence by stating that it is sometimes smaller, but often larger.

We rephrased the sentence for clarity, it now reads (lines 55-58):

“While the genomes of Octopodiformes (Octopus, Eledone, Argonauta) are either smaller than (1.1 Gigabases or Gb [45]) or comparable in size to that of humans (around 3 Gb [46,47]) the typical genomes of Decapodiformes (squids and cuttlefish) often reach 6 Gb [48,49].”

(4) L90: What tool was used to estimate the k-mer distribution of the long reads? Jellyfish? FastK? It's not mentioned anywhere in the text.

(5) L95: What k-mer size did the authors use to estimate k-mer distribution?

We thank the reviewer for pointing out this missing information, and have included the details in the methods (lines 692-694):

“The k-mer distribution was estimated using Meryl [165] within the Merfin [166] package with a k-mer size of 21, and genomeGenome size was estimated using GenomeScope [77] from Illumina short reads and PacBio HiFi data.”

(6) L99: What about using the most recent BUSCO databases? odb12?

We thank the reviewer for this question, which prompted us to compute BUSCO scores using the more recent odb12 database. The results are shown in Supplementary Figure 2C. Both gene sets have been refined by including more species and using a more stringent filtering approach, so the more recent database contains fewer and more conserved genes [4]. For the mollusca gene sets, a great improvement in completeness was observed between odb10 and odb12 (Supplementary Figure 2C); the metazoan completeness was marginally increased. Therefore, we evaluated all new assemblies produced since the first submission with the odb12 database.

(7) L107: How many scaffolds were obtained in total? After manual curation, how many of the scaffolds were placed in the "correct" chromosomes? How many scaffolds were in the shrapnel? Were these scaffolds mostly repetitive regions? Or did they contain important genetic information?

These are important questions. To evaluate the content of the “shrapnel”, we split the manually curated assembly into the 47 chromosomes and the 1840 residual scaffolds, and computed BUSCO scores for both. While the 47 chromosome scaffolds contain the majority of conserved genes: C:92.9%[S:92.7%,D:0.1%],F:4.0%,M:3.1% with metazoa_odb12 and C:88.7%[S:88.0%,D:0.7%],F:4.4%,M:6.9% with mollusca_odb12, the unplaced scaffolds still contain a few BUSCOs: C:2.5%[S:2.4%,D:0.1%],F:2.4%,M:95.1% from metazoa_odb12 and C:1.9%[S:1.7%,D:0.2%],F:1.2%,M:96.9% from mollusca_odb12. Even if only a few BUSCOs are present on these scaffolds, it means they contain important genetic information. Additionally, we observed low, but non-zero alignment of RNA reads to these scaffolds. We observed a slightly elevated repeat content in the unplaced scaffolds (Author response image 2), and a variable base composition (Figure 1C) compared to the chromosome scaffolds.

Author response image 2.

Quantification of repeat content in chromosome scaffolds and unplaced residual scaffolds. Density plot showing fraction of repeat masked bases in total sequence length for chromosome scaffolds (i.e. scaffolds 1-47) in teal and all remaining small scaffolds (1840 scaffolds) in purple. Median repeat fraction is shown as vertical lines.

The slightly elevated repeat content in the unplaced scaffolds provides a likely explanation for their fragmented state: repeat-rich regions are inherently difficult to assemble and scaffold, as repetitive sequences cause ambiguous read alignments that prevent contigs from being confidently joined or anchored to chromosomal scaffolds during HiC-based scaffolding. This is consistent with the near-complete absence of BUSCO genes from the unplaced scaffolds - not because these fragments lack biologically relevant sequence entirely, as evidenced by the residual BUSCO hits and RNA read alignments, but because the gene-rich portions of the genome are largely captured in the 47 chromosome scaffolds. The unplaced scaffolds instead likely represent fragmented contigs from repetitive or low-complexity genomic regions, such as centromeres, telomeres, and transposable element clusters, where assembly graph complexity and collapsed repeats prevent confident placement. The variable base composition further supports this interpretation, as GC-extreme or low-complexity sequences are disproportionately represented in assembly shrapnel. Together, these observations suggest that the unplaced scaffolds contain limited unique coding content but reflect genuine repeat-rich genomic sequence that cannot currently be placed without additional long-range information, such as optical mapping or ultra-long reads.

(8) L33, 53, 240, 255, 279: Decapodiformes, not in italics.

We changed this throughout the text.

(9) L228: Can you put this expansion in perspective with other taxa?

We added a more detailed comparison of our gene family expansion with different species to the revised manuscript, as detailed in response 4 to reviewer 1.

(10) L251: "However, our results show how difficult it still is to assemble large genomes with high karyotype numbers." Can you clarify how your results show this, because it is equally spectacular to assemble the karyotype with only PacBio and Hi-C data (and no linkage mapping).

Indeed, it is correct that the recent improvements in data quality and scaffolding algorithms enable these “spectacular” chromosome-scale assemblies without the need for linkage mapping. This sentence reflected our expectation to resolve a clear karyotype as has been demonstrated for multiple cephalopod genomes in recent years, including two cuttlefish species (Octopus bimaculoides, Octopus vulgaris, Euprymna scolopes, Euprymna berryi, Acanthosepion lycidas and Acanthosepion esculenta). To our knowledge, none of these publications used linkage mapping or cytogenetic methods to confirm the karyotype. In this light, our resulting chromosome number and the discrepancy to a second assembly of the same species led us to this conclusion. We updated the section in the revised discussion as follows (lines 466-473):

“Taken together, our results illustrate the difficulty of assembling large genomes with high repeat content and large karyotypes, at least from sequencing data alone. Internal validation methods and genome comparisons across species are therefore important. Convergence of reliable estimates will, in turn, help identify chromosomal fusion-with-mixing events (FWM; fusion of two ancestral chromosomes followed by extensive shuffling of their gene content) that are clade specific. Early branching order in Decapodiformes has been notoriously unstable [53,84,94,144–147]; thus, such rare and irreversible FWM characters could be useful in further phylogenetic analysis of this clade [51,148].”

(11) L419: Why use the phased haplotype 1 instead of the primary assembly generated by hifiasm?

We thank the reviewer for this important question. We used the phased haplotype assembly because it provides a biologically coherent representation with the least amount of duplication by avoiding allele-collapsing and haplotype-switching that can be present in the primary assembly. We reasoned that this would result in clearer gene models and a more accurate representation of structural variation. However, we acknowledge that this comes at the cost of reduced contiguity and completeness, as becomes apparent in our BUSCO comparison shown in Supplementary Figure 2, where the phased haplotypes have fewer duplicated genes than the primary assembly, but more missing genes in turn. When reassembling both datasets for our comparison, we used the primary assembly to use the longest contigs as input for scaffolding.

(12) L444: It is unclear from what tissues and life stages RNA-seq data were used or were available from other species.

This is an important detail. RNA-seq data was collected from two adult Sepia officinalis, from various tissues (whole brain, retina, skin, mantle, arm, tentacle). For the long-read PacBio Isoseq data, tissue was taken from the animal used for genome sequencing (6 months old), and tissue for short-read Illumina RNA-seq was taken from another adult (8 months old). The data have been released on SRA (study accession SRP570862), where all sample details are listed as well. We added the SRA accession to the data availability section of the revised manuscript. We clarified the relevant sections in the methods:

lines 628-629:

“RNA was isolated from various flash-frozen tissues (different brain areas, mantle/epidermis, arm/tentacle; 5-10 mg each).”

lines 678-680:

“For short-read RNA sequencing, tissue from another animal (8-month-old adult, F0 from eggs collected in Normandie, France) was used. RNA was isolated from various flash-frozen tissues (different brain areas, skin and retina; 5 mg each).”

(13) L454, 469: Why is minimap2 in italics? It wasn't formatted like this before. Same for StringTie.

We thank the reviewer for their detailed methods review. In the updated methods section, all formatting of used softwares was harmonized.

(14) L461: Lophotrochozoa is a clade, not a genus or species. Not in italics.

This is now changed throughout the revised manuscript.

(15) Figure 1D: Axes labels are hard to read.

We have now increased the axis label size.

(16) Figure 2: Consider increasing font sizes. Many chromosome orientations seem to be flipped across species, which makes it harder to see smaller-scale rearrangements or notice less conserved chromosomes. Would it make sense to standardize these?

We increased the font sizes and plotted only fully collinear syntenic blocks (instead of aggregated syntenic regions, the default of GENESPACE) for improved readability.

References:

Below are references cited in our responses. References from the reproduced manuscript sections are included in the revised manuscript.

(1) Secomandi, S., Gallo, G.R., Rossi, R., Rodríguez Fernandes, C., Jarvis, E.D., Bonisoli-Alquati, A., Gianfranceschi, L., and Formenti, G. (2025). Pangenome graphs and their applications in biodiversity genomics. Nat. Genet. 57, 13–26. https://doi.org/10.1038/s41588-024-02029-6.

(2) Open2C, Abdennur, N., Fudenberg, G., Flyamer, I.M., Galitsyna, A.A., Goloborodko, A., Imakaev, M., and Venev, S.V. (2023). Pairtools: from sequencing data to chromosome contacts. Preprint at bioRxiv, https://doi.org/10.1101/2023.02.13.528389 https://doi.org/10.1101/2023.02.13.528389.

(3) Lupše, N., Reid, A., Taite, M., Kubodera, T., and Allcock, A.L. (2023). Cuttlefishes (Cephalopoda, Sepiidae): the bare bones—an hypothesis of relationships. Mar. Biol. 170, 93. https://doi.org/10.1007/s00227-023-04195-3.

(4) Tegenfeldt, F., Kuznetsov, D., Manni, M., Berkeley, M., Zdobnov, E.M., and Kriventseva, E.V. (2025). OrthoDB and BUSCO update: annotation of orthologs with wider sampling of genomes. Nucleic Acids Res. 53, D516–D522. https://doi.org/10.1093/nar/gkae987.

The continuous downward flow of water symbolizes grief without resolution. Water disappears into a void that cannot be filled, rhetorically representing permanent loss. The repetition also creates meditative rhythm, encouraging contemplation rather than political interpretation.

https://www.researchgate.net/publication/354640770_Enshrining_Terror_for_the_Nation_Affect_and_Nationalism_at_the_National_September_11_Memorial_and_Museum

"The 9/11 and Vietnam Veterans Memorials: Interpreting Experience in the Landscape of Loss" https://ecommons.cornell.edu/entities/publication/0aa6f356-62e8-4eb8-ad4c-273dfebb5dce/full

"Making of the Vietnam Veterans Memorial" https://daily.jstor.org/the-making-of-the-vietnam-veterans-memorial/

love of wisdom

eclipsed the

Wisdom in Love

understand what is the case

antroposopia?

yes

absolute understanding

destroys what's human

local absolutes are real human spirit is one of those

very strong statement

The paper has now been published under the title "Quantifying phage-host dynamics using droplet microfluidics" in Nature Communications:

Givelet, L., von Schönberg, S., Katzmeier, F. et al. Quantifying phage-host dynamics using droplet microfluidics. Nat Commun 17, 3857 (2026). https://doi.org/10.1038/s41467-026-72427-3

eLife Assessment

This study presents valuable evidence of sex differences in oxycodone relapse-related behavior alongside novel characterization of synaptic adaptations in the paraventricular thalamus - nucleus accumbens shell circuit. The authors show that females exhibit heightened cue-induced seeking after 14 days, but not 1 day, of abstinence, while both sexes display similar time-dependent strengthening of paraventricular thalamus - nucleus accumbens shell glutamatergic transmission. The revised manuscript strengthens the work through improved statistical analyses, clearer interpretation, and expanded integration with prior literature. The strength of evidence is solid. However, association among experiments is incomplete, as the sex-specific behavioral effect is not reflected in circuit-level plasticity, and no causal manipulations test pathway involvement in relapse. Future work could link these circuit adaptations to sex-specific relapse vulnerability.

Reviewer #1 (Public review):

Summary:

This manuscript by Alonso-Caraballo et al, is a novel piece of work that examines the impact of oxycodone self-administration on neural plasticity within paraventricular thalamic (PVT) to nucleus accumbens shell (Shell) pathway - two regions shown to play a key role in cue-induced drug seeking on their own, and whether this plasticity varies based on abstinence period and biological sex.

Strengths:

The authors show using a clinically relevant long-access model of opioid self-administration promotes dependence and acute withdrawal in both male and female rats. During subsequent cue-induced relapse tests at 1 or 14-days following the conclusion of self-administration, data show that while both male and females demonstrate drug-seeking behavior at both time points, females show a further elevation in responding on day 14 versus day 1 that is not observed in the males. When accounting for past work showing elevations in drug seeking in males after 30 days, these data indicate that craving-induced relapse for opioids may develop faster and may be more pronounced in females compared to males.

These behavioral findings were paralleled by use of ex vivo acute slice electrophysiology and circuit-specific ex vivo optogenetics to examine the impact of oxycodone self-administration on synaptic strength within the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway(s). Data support a time-dependent but sex independent strengthening of glutamatergic signaling at PVT-to-NAcSh medium spiny neurons (MSNs) that is only present following a relapse test at 14 days post abstinence in males versus females, providing the first evidence that opioid self-administration and/or cue-induced drug-seeking augments this pathway. Using an extensive set of physiological measures, the authors show that this increased synaptic strength reflects a upregulation of presynaptic release probability. Further, this upregulation of excitatory signaling aligned temporally with an increase in MSN excitability, as assessed by increases in action potential firing frequency. Finally, the authors provide the first evidence that similar to other inputs to the NAcSh, PVT projections innervate both MSN as well as local interneurons, promoting a GABA-A specific feedforward inhibitory circuit. Interestingly, unlike direct excitatory inputs to MSNs, no changes were observed ostensibly within this feedforward circuit, highlighting a selective enhancement of excitatory drive and output of MSNs with protracted abstinence.

Overall, these data highlight a potential role for heightened synaptic strength within the PVT-NAcSh pathway in cue-induced relapse behavior during protracted abstinence and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals.

Weaknesses:

Overall, the experimental approach and data provided appear rigorous and support their overall conclusions and achieve their goal of understanding how opioid self-administration impacts synaptic strength within the PVT-NAcSh pathway. Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that reexposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

With regard to interpretation of electrophysiology findings, the lack of inclusion of an abstinence only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug induced adaptations in the NAc requires a post drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exopsure -- an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input-output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

Overall, these findings provide novel insight into how the PVT-NAcSh pathway is altered by opioid self-administration and whether this is unique based on abstinence period and sex. Importantly, these were the primary objectives stated by the author. Data highlight a potential role for the observed adaptations in relapse behavior and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals. However, it should be noted that no causal link is demonstrated without experiments to reduce/prevent relapse.

Comments on revisions:

The authors addressed previous concerns brought up, specifically by clarifying data interpretation as well as text modifications related to potential caveats of these interpretations. However, I recommend that the title be changed to not focus on sex differences to avoid misunderstanding. The authors should also address the lack of difference physiologically compared to the behavior as a caveat more clearly in the discussion (i.e. likely suggests this isn't the pathway driving the difference).

Reviewer #2 (Public review):

Summary:

This is an interesting paper from Alonso-Caraballo and colleagues that examines the influence of opioid use, acute and prolonged abstinence, and sex on cue-induced relapse and paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) medium spiny neurons circuit physiology. The study presents a valuable finding that following prolonged, but not acute abstinence from oxycodone self-administration, female rodents exhibit higher relapse rates to drug paired cues. Additionally, the study presents the useful finding that prolonged abstinence increased PVT-NAcSh MSN synaptic strength in both sexes, an effect that is likely due to presynaptic adaptations. While the evidence to support these two findings is solid, further experiments are required to determine the functional role of the PVT-NAcSh MSN circuit in relapse following prolonged oxycodone abstinence, and the mechanism underlying the heightened relapse vulnerability in females in this model of opioid use disorder.

Strengths:

The paper is interesting, well written and presented, and the experiments are well designed and conducted. The revised analysis of spike count data that models the hierarchical structure of the data is appropriate to overcome low animal numbers and the potential for oversampling. The authors are transparent in reporting the results related to this analysis in figure 5 and acknowledge the study is underpowered to confirm the trend of increased intrinsic excitability in male MSNs following prolonged oxycodone analysis.

Weaknesses:

A major weakness of this study is the disconnect between the behavioral and neurophysiological data reported. While a striking sex difference in relapse-like behavior is observed, there are no statistically significant sex differences in any of the neurophysiological data reported. Moreover, without an experiment to functionally test the role of the PVT-NAc projection in relapse-like behavior following prolonged oxycodone these two arms of the study seem divorced.

While the authors don't directly conclude that the PVT-NAc MSN circuit is required for relapse following prolonged oxycodone abstinences, in the introduction the authors state they aim to test the hypothesis that increased synaptic strength in PVT-NAcSh projections are necessary for drug-seeking. This study does not include the required experiments to test this hypothesis.

Impact:

The topic is of interest to the field of substance use disorders and gives solid evidence for the need to consider targeted therapeutics aimed at relapse prevention in opioid use disorder.

Reviewer #3 (Public review):

Summary:

Alonso-Caraballo et al. use behavioral testing and ex vivo patch-clamp electrophysiology combined with circuit-specific optogenetic stimulation of PVT terminals to examine how oxycodone self-administration and abstinence duration shape cue-induced relapse and PVT-NAcSh synaptic transmission in male and female rats. In the revision, the authors reanalyzed intrinsic excitability using nested hierarchical GLMMs, acknowledged the low power in the male prolonged-abstinence group, and expanded the discussion of relevant PVT-NAc literature. These changes improve the manuscript. That said, most of the revisions are textual and the main experimental gap remains. Both sexes show increased oxycodone seeking compared to saline at 14 days, but only females show a time-dependent incubation from 1 to 14 days, and the PVT-NAcSh synaptic strengthening is the same in both sexes. Nothing in the revision brings those two observations closer together. The excitability data also come from NAcSh MSNs with no confirmation of PVT connectivity, which limits what circuit-specific conclusions can be drawn. The study is a solid characterization of abstinence-related synaptic changes in this pathway, but some of the conclusions still go further than the data allow.

Strengths:

The behavioral characterization is thorough and well-executed, covering self-administration, somatic withdrawal, and cue-induced relapse across two abstinence durations in both sexes. The sex-specific escalation in oxycodone seeking from 1 to 14 days in females but not males is a clear and compelling finding. The use of circuit-specific ex vivo optogenetics to isolate PVT terminal inputs onto NAcSh neurons is a genuine methodological strength, and the demonstration of feedforward inhibitory recruitment through local GABAergic interneurons adds meaningful novelty to the circuit characterization. The reanalysis of intrinsic excitability using nested hierarchical GLMMs appropriately accounts for the non-independence of cells recorded within the same animal and is a real improvement over the original approach. The expanded discussion of prior PVT-NAc work, particularly the more accurate treatment of Keyes et al. (2020) and Paniccia et al. (2024), better situates the findings within the existing literature.

Weaknesses:

The core limitation of the study remains unchanged after revision. The PVT-NAcSh synaptic strengthening after prolonged abstinence is statistically indistinguishable between sexes, while females but not males show a time-dependent escalation in oxycodone seeking from 1 to 14 days of abstinence. The Discussion proposes hormonal modulation or differences in upstream inputs as possible explanations, but none of these are tested and the gap is left unresolved. The intrinsic excitability recordings come from NAcSh MSNs with no confirmation that those neurons receive direct PVT input, which was raised in the original review, acknowledged in the revision, and not experimentally addressed. The male prolonged-abstinence excitability trend has approximately 20% statistical power and is non-significant, yet the Discussion interprets it as a potential neuroadaptation that could facilitate signal flow through the PVT-NAcSh circuit and contribute to relapse, which goes well beyond what the data support. The failure to distinguish between D1 and D2 MSNs remains a significant limitation given that cell-type-specific plasticity at PVT-NAc synapses has been shown to be directly relevant to opioid seeking in prior work. Finally, the Conclusion builds a mechanistic framework around D2 MSNs, PV interneurons, and D1 MSNs that is drawn from studies using different drugs or experimental designs, and none of these cell-type-specific mechanisms are tested in the present experiments.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that re-exposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1-hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

We agree that the current design does not allow us to directly assess whether cue induced drug-seeking is augmented relative to the average self-administration intake. However, this comparison was not a question examined in the manuscript and was not an intended interpretation of the data. Our analyses and interpretations focused on comparisons between saline and oxycodone groups tested under identical cue-induced relapse conditions. While it does not change or contradict the reviewer’s point, we would also like to clarify that the relapse test was 2 hours long.

(2) With regard to the interpretation of electrophysiology findings, the lack of inclusion of an abstinence-only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test.

The inclusion of an abstinence-only control group would have been required to definitively dissociate synaptic changes driven by abstinence alone from those arising from an interaction between abstinence and re-exposure to the operant context during the relapse test. In the present study, electrophysiological recordings were intentionally performed 30 to 45 minutes following the relapse test to capture synaptic modifications associated with cue-induced drug-seeking after abstinence. Accordingly, we interpret these findings as reflecting the neural state following relapse rather than abstinence alone, and we have revised the text accordingly to clarify this point.

(3) With regard to the interpretation of electrophysiology findings, the lack of inclusion of an abstinence-only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug-induced adaptations in the NAc require a post-drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exposure - an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

We thank the reviewer for highlighting relevant literature showing that drug or cue re exposure can modify or reverse drug-induced plasticity in NAc-related circuits. We want to clarify that, in our dataset, synaptic changes in the PVT-NAcSh pathway are seen after 14 days of abstinence, but not after 1 day. Therefore, the lack of effect at the earlier time point and its appearance after extended abstinence support the idea of time-dependent plasticity. Although electrophysiological recordings were taken soon after the relapse test, this temporal pattern argues against relapse testing alone as the primary driver of the observed synaptic changes. We have updated the text to clarify this point.

(4) While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

We agree that unchanged AMPAR/NMDAR ratios and rectification index suggest against altered AMPAR subunit composition or simple postsynaptic sensitivity changes. Although receptor-specific input-output analyses would be necessary to definitively rule out proportional increases in both AMPA and NMDA receptor currents, we have updated the manuscript to clarify that our conclusions are limited to the synaptic measures we obtained. The revised text now states that acute or prolonged abstinence “might have no detectable postsynaptic effects as assessed by these synaptic measures” at PVT-NAcSh synapses.

Reviewer #2 (Public review):

(5) While this paper is certainly interesting, and well-written, and the experiments seem to be well performed, the behavioral and physiological effects observed are somewhat divorced. Specifically, what accounts for the heightened relapse in females? Since no opioid-related sex differences were observed in PVT-NAcSh neurophysiology, it is unclear how the behavioral and neurophysiological data fit together. Furthermore, the lack of functional manipulation of PVT-NAcSh circuitry leaves one to wonder if this circuit is even important for the behavior that the authors are measuring. I would be more positive about this study if the authors were able to resolve either of the two issues noted above.

A key challenge in circuit-based studies of motivated behavior is connecting circuit-level plasticity to complex, sex-dependent behavioral phenotypes. In this study, we do not mean to imply that synaptic plasticity within the PVT-NAcSh projection alone explains the increased relapse seen in females. Instead, our electrophysiological data indicate that this projection experiences time-dependent, abstinence-dependent changes in synaptic strength, offering important insights into when and where circuit-level adaptations may occur. We also believe that the lack of obvious sex differences in PVT-NAcSh synaptic strength does not rule out this circuit's role in sex-specific behavior. Growing evidence suggests that sex differences in relapse and motivated behaviors may stem from different modulation of shared circuits (for example, via ovarian hormones, neuromodulatory tone, or upstream inputs), rather than from significant differences in baseline synaptic properties within a given projection. Regarding circuit relevance, extensive previous research has identified the PVTNAcSh pathway as a critical regulator of cue-induced reward seeking and relapse. Our findings expand on this by showing that this projection displays abstinence-dependent synaptic strengthening after oxycodone self-administration. Although functional manipulation of this circuit is needed to confirm its causal role, such experiments were beyond the scope of this study.

(6) There are insufficient animals in some cases. For example, in Figure 4, the Male Saline 14-day abstinence group (n = 3 rats) has less than half of the excitability as compared to the Male Saline 1-day abstinence group (n = 7 rats). This is likely due to variance between animals and, possibly, oversampling. Thus, more rats need to be added to the 14-day abstinence group. Additionally, the range of n neurons/rat should be reported for each experiment to ensure readers that oversampling from single animals is not occurring.

We appreciate the reviewer's concern regarding the number of animals and the potential for oversampling. We take this concern seriously and have substantially revised our statistical approach in response.

All spike count data were reanalyzed using nested hierarchical Poisson generalized linear mixed-effects models (GLMMs), fitted separately for each sex and abstinence duration. Each model included injected current (mean-centered), drug condition, and their interaction as fixed effects, with random intercepts and slopes for injected current at the animal level, and random intercepts for cells nested within animals. Importantly, this reanalysis changed several of our original conclusions. Effects that appeared significant under the conventional cell-level analysis were no longer statistically significant once the hierarchical structure of the data was properly modeled. We report these corrected results transparently throughout the revised manuscript.

However, in males after prolonged abstinence, oxycodone-treated animals showed a higher spike output than controls, with a large effect size. Post-hoc analysis showed only 20% power with current sample (3 saline, 4 oxycodone rats). To reach 80% power, 13 rats per group are needed. We report this as a trend that warrants further study and have revised related sections to reflect this. The data suggest a possible neuroadaptation in males that the study is underpowered to confirm, not a null effect.

In response to this comment, we have updated Figure 5, the Results and Discussion sections, and the Statistics/Methods section to clearly describe the nested hierarchical modeling approach, report corrected statistical values, and acknowledge the power limitation for the male prolonged abstinence group. The figure legend now reports the number of neurons recorded per rat, showing the distribution across animals rather than individual subjects.

(7) The IPSC data, for example in Figure 4, is one of the more novel experiments in the manuscript. However, it is quite challenging to see the difference between males and females, saline and oxycodone, at low stimulation intensities within the graph. Authors should expand this so that reviewers/readers can see those data, especially considering other work suggesting that PVT synaptic input onto select NAc interneurons is disrupted following opioid self-administration. Additional comment: It's also interesting that the IPSC amplitude seems to be maximal at ~2mW of light, whereas ~11 mW is required to evoke maximal EPSC amplitude. It would be interesting to know the authors' thoughts on why this may be.

While visual separation between conditions at low light levels is subtle, we addressed this directly using linear mixed-effects modeling, which evaluates IPSC amplitudes across the full range of stimulation intensities while accounting for repeated measurements from cells nested within animals. This approach provides greater sensitivity than visual inspection alone and avoids over interpretation of noise at individual stimulation levels.

Using this framework, we observed robust main effects of light intensity in both males and females, indicating preserved recruitment of inhibitory synaptic responses as stimulation increased. Importantly, no significant Light × Condition interactions were detected in either sex, indicating that the scaling of IPSC amplitudes with light intensity was not altered by oxycodone exposure.

With respect to the observation that IPSC amplitudes appear to reach near-maximal levels at lower light intensities (~2 mW) compared to EPSCs (~11 mW), we agree that this distinction is intriguing. One possible explanation is that the depend on the recruitment of local interneurons. However, the number of interneurons activated by PVT interneurons is limited and inhibitory responses may reach a plateau at relatively low light intensities once these interneurons are fully recruited.

On the other hand, the increased intensity of photostimulation would result in an increase of monosynaptic EPSC amplitude over a wider range of stimulation (light) intensities, as increased intensity of light would recruit more ChR2-expressing PVT fibers, resulting in larger EPSCs.

(8) There is an inadequate description of what has been done to date on the PVT-NAc projection regarding opioid withdrawal, seeking, disinhibition, and the effects on synaptic physiology therein. For example, a critical paper, Keyes et al., 2020 Neuron, is not cited. Additionally, Paniccia et al., 2024 Neuron is inaccurately cited and insufficiently described. Both manuscripts should be described in some detail within the introduction, and the findings should be accurately contextualized within the broader circuit within the discussion.

In the revised manuscript, we expanded the Discussion to give a more thorough overview of previous research on the PVT-NAc pathway in relation to opioid-related behaviors and synaptic changes. Specifically, we added more detail about Keyes et al., 2020 and Paniccia et al., 2024, clarifying their findings and placing them within the context of the circuit mechanisms studied in our work. We also revised the text to ensure the descriptions of these studies are accurate and that their conclusions are properly related to our findings.

(9) Related to the above, the authors should provide a more comprehensive description of how PVT synapses onto cell-type specific neurons in the NAc which expand beyond MSNs, especially considering that PVT has been shown to influence drug/opioid seeking through the innervation of NAc neurons that are not MSNs. For example, see PMIDs 33947849, 36369508, 28973852, 38141605.

In the revised manuscript, we expanded the Discussion to describe the diversity of PVT projections within the NAc and the potential role of non-MSN neuronal populations in drug-related behaviors. We added discussion on the broader circuit context and other cell types where relevant to the focus on synaptic transmission onto MSNs. Since our experiments specifically examined synaptic physiology in MSNs, we focused the literature discussion on studies most directly related to MSNtargeted PVT inputs and opioid-related behaviors.

Reviewer #3 (Public review):

(10) Additional experiments could strengthen the results and help clarify synaptic mechanisms underpinning behavioral sex differences.

We agree that additional experiments focused on identifying cell-type-specific mechanisms within the PVT-NAcSh circuit would further enhance understanding of the neural substrates behind the observed behavioral sex differences. In the revised manuscript, we have expanded the Discussion to explicitly acknowledge these limitations and clarify the scope of our current study. Specifically, we discuss the possibility that sex-specific adaptations might occur in particular neuronal subpopulations or circuit components that were not resolved in the present experiments. We also mention that future research using cell-type–specific approaches will be necessary to determine if such mechanisms contribute to the increased oxycodone seeking seen in females after prolonged abstinence. We appreciate the reviewer’s suggestions and have incorporated this perspective into the revised manuscript to better contextualize our findings and outline future directions.

eLife Assessment

This study investigates the role of the Z-disc protein Zasp52 in Drosophila flight muscles and provides evidence that an intrinsically disordered region (IDR) helps to stabilize and promote the localization of the protein to the Z-disc. Overall, this represents an important study that provides insights into Z-disc function and maintenance. The data are convincing, supported by strong genetic evidence and behavioral tests, well-controlled experiments, and detailed statistical analyses. Additional functional analyses designed to tease out specialized regions within the newly described isoform of Zasp52 would further strengthen models regarding the function of the protein.

Reviewer #1 (Public review):

The manuscript by Ho and Schock investigates the role of the Z-disc protein Zasp52 during Drosophila flight muscle development. It was known before, mainly by findings from this group, that Zasp52 is required for normal sarcomere morphogenesis, specifically Z-disc morphogenesis in indirect flight muscles. But the exact molecular mechanism by which Zasp52 contributes, apart from the fact that it is localised there and is somehow involved in multimerization/cross-linking, was not clear. This paper proposes that an intrinsically disordered region (IDR) in Zasp52 is needed for some of its functions, by stabilising Zasp52 localisation at the Z-disc. Specifically, the IDR in Zasp52 is proposed to be required for Z-disc maintenance during the mechanical challenges of flight, while being dispensable for the initial morphogenesis during development. This hypothesis is supported by strong genetic evidence and behavioural tests, deleting Zasp's IDR impairs flight from mid-age onwards, while a block in flight activity lifts the phenotype.

However, some of the phenotypic analysis, in particular the bending of the sarcomere, likely upon mechanical challenge by muscle contractions, needs more detailed investigations to be fully convincing.

Strengths:

(1) The linker in the alternatively spliced exon 15 of Zasp52 was deleted with a state-of-the-art genetic editing strategy. Surprisingly, flies are homozygous viable, showing that this long part of the Zasp52 protein is not essential for animal survival or sarcomere morphogenesis.

(2) The observed sarcomere phenotypes with age, especially the bending Z-discs, are new and exciting.

(3) The displayed EM images document interesting phenotypes.

(4) Most of the observed phenotypes can be rescued by re-expression of the long Zasp52 isoform, which does contain the IDR region, but not by a shorter one without it, suggesting that IDR is important.

(5) FRAP data measure the local turnover of a short-ZaspGFP and show that this increased in the Zasp mutant lacking the IDR domain, suggesting that Zasp-IDR might stabilise Zasp at the Z-disc.

(6) Interestingly, flight and sarcomere morphology phenotypes can be rescued by preventing the flies from flying, suggesting that they are mechanically induced.

Weaknesses:

(1) The western blot quantifications of Zasp isoform expression are weak. No error bars are indicated in the quantifications; the quantifications appear to be more qualitative than quantitative. According to band intensities, the long Zasp isoforms seem to be less present compared to the shorter ones, even in the flight muscles.

(2) The phenotypic analysis of the sarcomere appears somewhat superficial throughout the paper. Only Zasp52 and phalloidin are shown; no other Z-disc or thick filament proteins. At least myosin stainings and overview images are important to better judge the phenotypic variations. Are the variants between individuals or regional in the same muscle?

(3) EM images would benefit from better quantification.

(4) Other proteins were not analysed with the FRAP-based turnover assay for comparison in wild type and mutant. All Z-proteins might turn over faster in the mutant with the defective Z-disc.

Reviewer #2 (Public review):

Summary and Strengths:

This in-depth genetic analysis of Zasp52 function in Drosophila indirect flight muscle (IFM) provides an interesting perspective regarding the role of a partially disordered region (IDR) in exon 15e. This exon seems to be exclusively present in IFM and contributes to the prevention of myofibril disintegration during aging, likely due to interactions of this region with Z-disc insertion and/or stability. The addition of an isoform (PR) that lacks exon 15e serves as a nice control to illustrate the necessity of exon 15e in muscle structure and function. Overall, the manuscript is exceptionally well-written, logical, with nicely controlled experiments and detailed statistical analysis that largely support the conclusions drawn by the authors. While exon 15e is clearly involved in preventing muscle degeneration, a solid role for thin filament stability is not clearly shown (as mentioned in the abstract). In addition, which regions/how the proteins of the IDR may contribute are unclear.

Weaknesses:

(1) It is not clear in Figure S1A where exon 15e fits within the Zasp52 locus schematic. This is important as a premise of this paper describes this region to be key, and proof from multiple prediction programs would lend more weight to the prediction of the exon being largely disordered. Inclusion of the discussed short linear motifs, comparison with Canoe or LBD3 for similarities and/or an Alphafold structure would help make the authors' point (colorized with known domains).

(2) Interesting that immobilization rescues the deterioration phenotypes. The authors should explain in more detail how this was done to avoid dehydration/starvation of the flies.

(3) There is a lot of discussion about the potential function of the IDR region, specifically a putative actin binding motif or other 'ordered' regions that may contain short linear motifs. It would strengthen the findings to show which of these may be essential for Zasp52 function in the IFM. The ability to bind actin could be tested biochemically, and/or smaller deletions could be made to unequivocally test the role of the ABD vs other predicted motifs using genetics. If some of these regions are more ordered, where do they lie within, and do they form a predicted fold or structure that gives insight into function?

Author response:

Public Reviews:

Reviewer #1 (Public review):

The manuscript by Ho and Schock investigates the role of the Z-disc protein Zasp52 during Drosophila flight muscle development. It was known before, mainly by findings from this group, that Zasp52 is required for normal sarcomere morphogenesis, specifically Z-disc morphogenesis in indirect flight muscles. But the exact molecular mechanism by which Zasp52 contributes, apart from the fact that it is localised there and is somehow involved in multimerization/cross-linking, was not clear. This paper proposes that an intrinsically disordered region (IDR) in Zasp52 is needed for some of its functions, by stabilising Zasp52 localisation at the Z-disc. Specifically, the IDR in Zasp52 is proposed to be required for Z-disc maintenance during the mechanical challenges of flight, while being dispensable for the initial morphogenesis during development. This hypothesis is supported by strong genetic evidence and behavioural tests, deleting Zasp's IDR impairs flight from mid-age onwards, while a block in flight activity lifts the phenotype.

However, some of the phenotypic analysis, in particular the bending of the sarcomere, likely upon mechanical challenge by muscle contractions, needs more detailed investigations to be fully convincing.

Strengths:

(1) The linker in the alternatively spliced exon 15 of Zasp52 was deleted with a state-of-the-art genetic editing strategy. Surprisingly, flies are homozygous viable, showing that this long part of the Zasp52 protein is not essential for animal survival or sarcomere morphogenesis.

(2) The observed sarcomere phenotypes with age, especially the bending Z-discs, are new and exciting.

(3) The displayed EM images document interesting phenotypes.

(4) Most of the observed phenotypes can be rescued by re-expression of the long Zasp52 isoform, which does contain the IDR region, but not by a shorter one without it, suggesting that IDR is important.

(5) FRAP data measure the local turnover of a short-ZaspGFP and show that this increased in the Zasp mutant lacking the IDR domain, suggesting that Zasp-IDR might stabilise Zasp at the Z-disc.

(6) Interestingly, flight and sarcomere morphology phenotypes can be rescued by preventing the flies from flying, suggesting that they are mechanically induced.

Weaknesses:

(1) The western blot quantifications of Zasp isoform expression are weak. No error bars are indicated in the quantifications; the quantifications appear to be more qualitative than quantitative. According to band intensities, the long Zasp isoforms seem to be less present compared to the shorter ones, even in the flight muscles.

We will work on including quantifications with error bars for the Western blots in our resubmission. It is important to keep in mind that the main point in figure 1B is that there are plenty of exon15e-containing isoforms in IFM, in contrast to other tissues with very limited exon15e-containing isoforms. This is confirmed by the analysis of RNAseq data in figure 1C, and of course, by the flightless phenotype of the exon15e mutant.

(2) The phenotypic analysis of the sarcomere appears somewhat superficial throughout the paper. Only Zasp52 and phalloidin are shown; no other Z-disc or thick filament proteins. At least myosin stainings and overview images are important to better judge the phenotypic variations. Are the variants between individuals or regional in the same muscle?

Our images are representative of the observed phenotypes. We aim to provide overview images and other stainings to better illustrate the phenotypic variations in the revised version. Phenotypes are consistently present across all individuals, as reflected in our replicates. Interestingly, they appear to not be randomly interspersed among the sarcomeres but concentrated in certain regions of muscle more than others.

(3) EM images would benefit from better quantification.

We do not believe that EM images can be meaningfully quantified, because of the many selection steps preceding image acquisition.

(4) Other proteins were not analysed with the FRAP-based turnover assay for comparison in wild type and mutant. All Z-proteins might turn over faster in the mutant with the defective Z-disc.

This is the point we are trying to make. The Zasp52 IDR acts like a glue stabilizing all Z-disc proteins. We performed this experiment as a first step to explore whether an exon15e-lacking system exhibited modified dynamics, and we aim to provide more data in the revised version.

Reviewer #2 (Public review):

Summary and Strengths:

This in-depth genetic analysis of Zasp52 function in Drosophila indirect flight muscle (IFM) provides an interesting perspective regarding the role of a partially disordered region (IDR) in exon 15e. This exon seems to be exclusively present in IFM and contributes to the prevention of myofibril disintegration during aging, likely due to interactions of this region with Z-disc insertion and/or stability. The addition of an isoform (PR) that lacks exon 15e serves as a nice control to illustrate the necessity of exon 15e in muscle structure and function. Overall, the manuscript is exceptionally well-written, logical, with nicely controlled experiments and detailed statistical analysis that largely support the conclusions drawn by the authors. While exon 15e is clearly involved in preventing muscle degeneration, a solid role for thin filament stability is not clearly shown (as mentioned in the abstract). In addition, which regions/how the proteins of the IDR may contribute are unclear.

Weaknesses:

(1) It is not clear in Figure S1A where exon 15e fits within the Zasp52 locus schematic. This is important as a premise of this paper describes this region to be key, and proof from multiple prediction programs would lend more weight to the prediction of the exon being largely disordered. Inclusion of the discussed short linear motifs, comparison with Canoe or LBD3 for similarities and/or an Alphafold structure would help make the authors' point (colorized with known domains).

We will add a bar below figure S2A to show the region corresponding to exon 15e. We used three disorder prediction programs and one structure (order) prediction program. The majority of exon15e is completely disordered and of very low confidence score, and thus uninformative to display as an Alphafold structure. Likewise, IDR’s are very difficult to classify, therefore we cannot say much more than that LDB3, Zasp52, and Canoe contain IDRs, with Zasp52 and Canoe both having an actin-binding domain within the IDR. We will provide more data on the function of the ABD in the revised version.

(2) Interesting that immobilization rescues the deterioration phenotypes. The authors should explain in more detail how this was done to avoid dehydration/starvation of the flies.

We will provide more details in the revised version.

(3) There is a lot of discussion about the potential function of the IDR region, specifically a putative actin binding motif or other 'ordered' regions that may contain short linear motifs. It would strengthen the findings to show which of these may be essential for Zasp52 function in the IFM. The ability to bind actin could be tested biochemically, and/or smaller deletions could be made to unequivocally test the role of the ABD vs other predicted motifs using genetics. If some of these regions are more ordered, where do they lie within, and do they form a predicted fold or structure that gives insight into function?

We will provide data on the function of the ABD in the revised version.

Met-level extensibility as in OHS

a personal digital archive, to store and use whatever content the learner/knowledge worker need to gather, organize, explore, reuse in a form that meets personal needs, and produce artifact in a form that she specifies, to harness personal creativity to personal problem solving

!4 Conceptipedia

This is a good accessibility point as it presents how important simple language is. Utilizing simple words makes it easier for users to understand, especially those with reading disorders, short attention spans, memory challenges or those with poorer digital literacy. This form of simple, clear writing ensures the web content is much more accessible to a wider range of users.

This is a link with descriptive wording and is a good example of an accessible link as the link very clearly discusses where it will take them. As opposed to it saying "click here" it tells you where you will be redirected, naming the exact source; The Web Content Accessibility Guidelines (WCAG) 2.0. This helps all users, and the screen readers specifically to understand the link they click prior to opening it. This connects to this weeks theme as accessibility is not solely about internet access, it also is about making online information accessible and usable for those with various needs.

This statement relates to web accessibility as it has content that is not just about design, but also discusses how someone can understand and use the information they learn. This is a very useful tool as it considers all readers, and considers those with unique differences such as any physical or cognitive disabilities.

This is a strong accessibility feature that utilizes a clear heading to organize the information neatly so it is easy to follow. This helps readers see when what they are reading and where exactly they are in the paper. This is especially useful for those who are looking for a specific piece of information as it can save time and help map out the page.

This is a table of contents which is often at the beginning or the very end of a paper, and is a very crucial accessibility feature as it helps users navigate the paper easily, saving time and allows for ultimate efficiency as it eliminates the need to scroll endlessly. This is especially useful for those who struggle with large amounts of information all at once.

eLife Assessment

This important study identified Mex3a protein with dual RNA-binding protein/ubiquitin ligase function as a pivotal regulator of olfactory sensory neurons (OSN) differentiation and lineage fidelity. The authors employed a combination of systems biology approaches (e.g., single-cell RNA sequencing, proteomics) and newly developed animal models (e.g., HyperTRIBE) to provide solid evidence that abrogation of Mex3a disrupts cilia structure and polarity of OSNs. Notwithstanding that this article is of a broad potential interest across different biomedical disciplines ranging from RNA to developmental biology, additional mechanistic data connecting identified Mex3a mRNA targets and ensuing OSN phenotypes would further strengthen this study.

Reviewer #1 (Public review):

The study by Escamilla del Arenal et al. utilized a conditional knockout mouse model to study the role of Mex3a in immature olfactory sensory neurons (OSN). Mex3a is a dual-functional protein that has RNA-binding function and ubiquitin-E3 ligase activity. The results revealed that Mex3a expression is critical for proper OSN differentiation and contributes to cell surface protein trafficking and translation, cilia structure, and planar cell polarity in mature neurons. Moreover, Mex3a enforces lineage fidelity, selectively repressing sustentacular programs in neurons and neuronal programs in sustentacular cells.

In addition, the authors established an in vivo HyperTRIBE mouse model to identify Mex3a RNA targets and incorporated UbiFast into the Mex3a conditional knockout (cKO) model to find its protein targets to investigate how Mex3a regulates OSN differentiation. The experimental systems are laborious and comprehensive, which allowed the authors to identify new Mex3a putative targets in OSN.

The phenotypic results derived from the conditional Mex3a cKO mice are solid. Mechanistic findings also revealed that, in addition to facilitating protein degradation, Mex3a may confer K27 ubiquitin linkage on its target proteins, which has a non-proteolytic role but affects target protein activity, other post-translational modifications, or protein-protein interactions. However, among all Mex3a putative targets, the authors decided to emphasize on the Mex3a-mediated K27 ubiquitination on stress granule protein Serbp1 and ribosome protein Rps7, and the association between Mex3a expression and Serbp1 and p-eEF2 ribosome recruitment. This Mex3a-Serbp1-p-eEF2 ribosome recruitment axis, although it can be important in Unfolded Protein Response (UPR) signaling, seems rather general and cannot explain the striking lineage-specific phenotypes observed in the mouse model. The authors need to provide more solid evidence to demonstrate that K27-Ubiquitinylation of Serbp1 is a key step of Mex3a function in OSN differentiation to strengthen the relation between the phenotypes and mechanism presented in this study.

Reviewer #2 (Public review):

Summary:

In this manuscript, Arenal and colleagues demonstrate that loss of Mex3a leads to defects in cell surface protein trafficking, translation, ciliary structure, and planar cell polarity in mature neurons. Through proteomic analyses, the authors show that Mex3a depletion alters the abundance of proteins involved in vesicular transport, lipid metabolism, and ribosome biogenesis. Using the HyperTRIBE approach, the authors further identify targets of Mex3a and provide evidence supporting a role for K27-linked ubiquitination in regulating these substrates. Mechanistically, the study suggests that Mex3a levels influence the recruitment of SERBP1 and phosphorylated eEF2 (p-eEF2) to ribosomes, contributing to translational repression.

Strengths:

Overall, this is a very interesting and well-written manuscript that significantly advances our understanding of Mex3a function and its role in neuronal development, particularly in olfactory sensory neurons. The data are clearly presented and thoughtfully interpreted.

Weaknesses:

I have a few minor comments that may further strengthen the manuscript and improve its accessibility to a broader readership.

(1) In Figure 3B, the authors describe Mex3a localization to cytoplasmic granules. However, it is unclear how these compartments were defined. It would strengthen the conclusions if the authors included co-localization experiments using established cytoplasmic granule markers (e.g., stress granule markers) to define the identity of these structures more precisely. This would clarify whether Mex3a associates with stress granules, RNA processing bodies, or another class of ribonucleoprotein granules.

(2) Functional validation of K27-linked ubiquitination on SERBP1<br /> To further define the functional significance of K27-linked ubiquitination, it would be informative to mutate the relevant lysine residue(s) on SERBP1 and examine whether this alters its recruitment to ribosomes or affects translational repression. Such an experiment would provide more direct evidence that K27-linked ubiquitination of SERBP1 mediates the observed translational effects.

(3) Discussion of vesicular trafficking and lipid metabolism targets<br /> The identification of Mex3a targets involved in vesicular trafficking and lipid metabolism, including COPII coat components such as Sec31a and lipid regulatory proteins such as Sec14 and PIP5K1A, is particularly intriguing. The authors may wish to expand the Discussion to address how regulation of these proteins could contribute to defects in plasma membrane trafficking and planar cell polarity. Integrating these findings with the observed cell surface trafficking phenotypes would further enhance the mechanistic framework of the study.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors investigate the role of the KH and RING domain-containing protein Mex3a in the differentiation and maturation of olfactory sensory neurons. Using conditional knockout of Mex3a in immature neurons, they show that mature olfactory sensory neurons display defects in membrane protein trafficking, including olfactory receptors and Adcy3, together with abnormalities in ciliary radial organization and planar cell polarity. Through single-cell RNA sequencing and quantitative proteomics, the authors further show that Mex3a-deficient neurons fail to properly resolve the unfolded protein response and exhibit transcriptomic features suggestive of lineage mixing with sustentacular cells. The study also introduces a methodological advance by adapting HyperTRIBE for use in transgenic mice, which enables the identification of in vivo Mex3a RNA targets, including components of Wnt signaling that appear to be under translational repression by Mex3a. The authors then pursue one of these targets to further explore the role of Mex3a in translational repression.

Strengths:

First, it addresses an important biological and conceptual question. Mex3a is a multifunctional protein with the potential to couple RNA regulation, protein homeostasis, and key cellular processes, yet its in vivo role in neuronal differentiation remains poorly understood. By focusing on Mex3a in olfactory sensory neurons, the manuscript asks a timely and important question of how post-transcriptional regulation contributes to the maturation of highly specialized neurons, including the establishment of ciliary architecture, membrane protein trafficking, and cell polarity. Second, the generation and validation of an inducible in vivo mouse HyperTRIBE system represents a technical advance. By incorporating the Adar deaminase domain into a transgenic mouse model, the authors establish a rigorous and useful approach for identifying Mex3a RNA targets in vivo, which is likely to be valuable to the wider RNA biology community. Third, the study integrates the Mex3a knockout model with single-cell RNA sequencing, quantitative mass spectrometry-based proteomics, ubiquitin profiling, and ribosome-related analyses, providing a broad and multilayered view of the Mex3a knockout phenotype. Finally, the imaging analyses revealing altered ciliary content and organization in olfactory sensory neurons identify an interesting and potentially important link between Mex3a, cilia biology, and vesicular trafficking. More broadly, the manuscript reflects a very substantial experimental effort, and each individual dataset has the potential to be useful for the field.

Weaknesses:

A main weakness of the manuscript is that the mechanistic links between the major findings remain somewhat correlative, and the biological narrative is not fully sustained through the later figures. The study documents defects in membrane trafficking, ciliary radial organization, and planar cell polarity, and it identifies candidate targets with clear relevance to these processes, including factors linked to vesicle trafficking. However, the manuscript then shifts its mechanistic focus toward translational regulators such as Serbp1 and Rps7, without adequately connecting these later analyses back to the core phenotypes established earlier. As a result, there is a noticeable disconnect between the phenotypic emphasis of the study and the mechanistic validation that follows.

A second weakness is that, given the breadth and potential importance of the datasets generated, validation remains limited for several of the major conclusions. This reduces confidence in the interpretation of the single-cell, proteomic, ubiquitin-related, and ribosome-associated analyses, and also limits the future value of these datasets as a resource for the field. Because the manuscript aims to address several major questions at once, stronger validation and clearer integration across the different experimental arms are needed for the conclusions to feel fully supported.

Finally, the HEK293T overexpression experiments are less solid than the in vivo analyses and do not provide equally strong support for the proposed mechanisms. In this context, some of the observed effects on cytoskeletal organization, membrane-less granule formation, and ribosome profiles may be indirect, which makes it difficult to weigh these findings alongside the much stronger in vivo phenotypes.

DOI: 10.1126/sciadv.adw4064

Resource: RRID:MMRRC_049020-UCD

Curator: @AleksanderDrozdz

SciCrunch record: RRID:MMRRC_049020-UCD

What is this?

DOI: 10.1180/gbi.2026.10007

Resource: University of Delaware Center for Bioinformatics and Computational Biology Core Facility (RRID:SCR_017696)

Curator: @scibot

SciCrunch record: RRID:SCR_017696

What is this?

Dear BioRxiv staff, This preprint is now published in Sci Adv since the 24th of April. DOI: 10.1126/sciadv.adz6211 Could you please updete it? Thanks for the continuous support to open science. Best, María Gómez

there is excellence without mathematics

Instead of separating schema in a DTD

write/compose documents with

explicitely named structural elements that are themselves selfcontained description of the role, function, purpose, affordance of the named element

Literally, a grove = is a Graph Representation Of property ValuEs. -

Groves provide constructs for describing directed graphs

consisting of - nodes, - which have properties, and - arcs, which connect the nodes.

The basic idea is that any data, especially structured documents, can be modeled using directed graphs.

A tree structure is a type of directed graph, and anyone familiar with parsing structured documents, be they XML documents or source code, will understand the utility of tree structures for representing those documents.

In the early 2000s Engelbart and a group of collaborators were coülaborating on the inception and creation of an Open HyperDocument System

formed a Launch Community for the same

The work we do in IndyLab builds on and aims to realize the vision. "The Future inherent certainty" that there could be (as the Wiki demonstrated) a simplest possible way that it could possibly work that would augment human interintellect via unbounddd, unenclosable, interpersonal co-laboration on the Web weaving the IndyWeb of Open collaborative HyperDocument system, what it wanted to be in the first place

Armed with the Pratcial Magic of Content Address-ability

aiming for

• human centered/meaningful
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articulations - auto poietic - eventually effective - formulations

with mutual arising - intellectual<br /> - intentional - transparency

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do the work win an/innotaation

so that what is of interest can be shared with others through a higher bandwidth

and opening a sa(l|p)ience scape for engagement , threaded linked conversations

connecting people interests and dieas

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record the salience and the associated ideas

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Property sets

let you define nodes, - which essentially are a set of properties. - These properties have names and a data type. Data types can either be primitive, familiar types such as strings and characters and integers, or they can be nodal -- another node, a list of nodes, or a node map (known in property set parlance as a named node list). Represented graphically, a node and its nodal properties are connected to each other by an arc, with the direction of the arc pointing towards the property

In order to describe the structure of a grove, you need a data modeling language. Property Sets are that language. They consist of an SGML DTD used to define the schema for a grove. I won't describe the property set syntax in detail here, as the ISO specification does a good job of documenting it.

Both Example 1 and Figure 1 should look familiar to computer programmers, and for good reason. One of the programmer's primary jobs is data modeling -- figuring out a way to represent data so that the software can manipulate it appropriately, and then expressing that data abstraction in a programming language. All programming languages, in fact, are data modeling languages, since they provide a way to express data models.

What exactly does this mean? At the lowest level, all data represented by a data model is addressable at any granularity, since software can address and retrieve any chunk of data stored in an internal data structure. The value of groves is that it provides an explicit way of specifying the smallest units of data that can be addressed by an application and a navigational scheme for describing the location of this data.

data model vs

salience scape

sapience scape

As a data modeling language, groves * fall between the two extremes, - leaning a bit towards the lower- level languages**.

The intent of groves is to enable addressability of data -

-- in other words, provide the capability to - describe unequivocally the location - of a chunk of data =

Towards the opposite end of the extreme, you have

data modeling languages such as - E/R and - UML diagrams,

which are more human-centered and

present a higher-level abstraction of the data.

The cost of these abstractions is the - lack of information that may be valuable in - implementing these data models.

For example, E/R diagrams allow you to specify

the cardinality of a relationship - (i.e. one-to-n, n-to-one, n-to-n),

but they do not allow you to express other kinds of constraints, and

they have no notion of primitive data types.

Consequently, they are not adequate to - generate equivalent source code - automatically and - completely,

although they could be used to generate stub code.

As a result, the data modeling part - of a programming language tends to be

close to the machine,

describing low-level structures in a way that - sometimes obscures the higher-level meaning of

a data model.

The intent of a programming language is for - the expression of a data model - (i.e. source code) to be translated into a - machine-readable representation of the data.

The casual tone in this part is really helpful since citation usually gets talked about in a pretty formal way. Viewing citation as a handy tool for staying organized and doing research down the line makes it seem way more practical and useful.

This section describes research as a collaborative process rather than as something completed entirely by an individual. This perspective illustrates why it is crucial to acknowledge outside sources and viewpoints in academic work.

This sentence clearly explains something many still get confused about during research projects. Including a short example showing the difference between quoting, paraphrasing, and summarizing could make this section even more useful for beginners.

t's seriously important to recognize that applying research completed by others to our own work can be challenging. The breakdown of honesty, respect, and responsibility is helpful because it links academic integrity to professional and ethical behavior outside the classroom. This section explains these concepts in a practical manner, rather than using overly technical language.

Gives academic citation more meaningful than simply avoiding plagiarism. The focus on clarity and transparency helps explain why citations are important within academic conversations and research.

This stood out because though out academic seetings citation is often taught only as a formatting requirement instead of explaining why it matters academically. The section does a good job connecting citation practices to communication, credibility, and academic integrity.

This section effectively explains how information shared across platforms online can quickly lose important context. The discussion about tracing claims, quotes, and media back to their original sources is particularly relevant in today's fast-paced information landscape.

This clarification is helpful because it highlights that citations are not just about support or approval. It encourages us to recognize that sources can be cited for various purposes, including criticism, comparison, and fostering meaningful discussions within academic research. This understanding can enhance the depth and quality of our work.

The explanation of lateral reading was particularly helpful because it offers a practical strategy rather than simply instructing readers to “check credibility.” Including a step-by-step example of a real lateral search could make this process even easier for beginners to understand. This also made me think about how AI's use in fact-checking isn't always accurate.

This section is noteworthy because skepticism is often seen in a negative light. However, it emphasizes that skepticism is a vital component of critical thinking and evaluating sources. The wording clarifies that assessing credibility involves asking thoughtful questions rather than simply distrusting everything automatically.

R0:

Reviewer #1: Recommendation 1: The modeling approach is fundamentally sound and relevant to TB control (a major public health issue), but the manuscript needs to address transparency (data sources, parameter justification) and clarify some methodological assumptions (e.g. definition of Reproduction number) before it can be accepted.

Recommendation 2: The sensitivity analysis methods (Pearson, PRCC) are suitable but need more rigor in reporting (sample details, uncertainty). The data-fitting procedure requires complete description (data source, fitting algorithm, goodness-of-fit). The authors should include more quantitative statistical assessment of their model fit and uncertainty in parameter effects.

Recommendation 3: The authors should provide a Data Availability Statement with actual access information (URL, DOI, or supplementary files) for the epidemiological data and any other material used. At the end of the file, an editorial link points to the Bangladesh NTP Annual Report 2022, which presumably provided TB case counts, but this is not an open dataset citation. Without accessible data, readers cannot reproduce or verify the results.

Recommendation 4: The writing requires major language editing. Grammatical issues and awkward phrasing occur repeatedly. Several citations are incomplete (marked as “[?]”). These issues hinder readability. The manuscript would benefit from careful editing by a fluent English speaker. With editing, the intellectual content is conveyable, but in its current state the language detracts from clarity.

Reviewer #2: This manuscript presents a mathematical modeling study on tuberculosis transmission using an SEITR framework incorporating distancing and treatment control strategies. The authors apply optimal control theory, stability analysis, sensitivity analysis, and data fitting to assess the impact of interventions. The topic is relevant to public health and epidemiological modeling, and the integration of control theory with real data is a valuable contribution. Overall, the study demonstrates potential scientific merit; however, several important issues should be addressed to improve the quality, clarity, and reliability of the work.

Major Strengths

The manuscript develops a structured SEITR model that includes exposed and treated compartments, allowing for a more realistic representation of tuberculosis dynamics.

The application of Pontryagin’s Maximum Principle to determine optimal intervention strategies is appropriate and well motivated.

The use of sensitivity analysis, including Pearson correlation and PRCC, strengthens the interpretation of key parameters.

Incorporation of real tuberculosis data from Bangladesh and curve fitting enhances the practical relevance of the study.

The findings highlight the importance of combined distancing and treatment strategies, which is relevant for policy planning.

Major Concerns

Language and Presentation The manuscript contains numerous grammatical errors, awkward phrasing, and unclear sentences that affect readability. Several sections require substantial English language editing. Professional proofreading is strongly recommended to improve clarity and coherence.

Incomplete and Missing Citations Many references in the text are marked with “[?]”, indicating missing citations. These must be replaced with appropriate and verifiable references. Failure to do so weakens the scientific credibility of the manuscript.

Reference Quality and Formatting Several references appear incomplete, inconsistently formatted, or potentially inaccurate. All references should be verified and formatted according to journal guidelines. The authors should ensure that all cited sources are reliable and accessible.

Mathematical Notation and Consistency Some notations are used ambiguously, particularly the symbol λ, which represents both the force of infection and adjoint variables. This may confuse readers. The notation should be revised for clarity. In addition, some derivations in the optimal control section are not sufficiently detailed and require clearer explanation.

Data Availability and Reproducibility The Data Availability Statement is insufficient. PLOS requires full access to underlying data and, where applicable, code. The authors should provide the dataset in an accessible repository and include links. Details on parameter estimation and model fitting procedures should also be expanded.

Model Validation While curve fitting is presented, quantitative goodness-of-fit measures (e.g., RMSE, R², confidence intervals) are not provided. Including these metrics would strengthen the validation of the model.

Overstatement of Novelty The novelty section contains strong claims that are not fully supported by the analysis. The authors should moderate these statements and clearly specify how their approach differs from and extends existing studies.

Figures and Visualization Some figures lack sufficient resolution, clear legends, and readable labels. All figures should be revised to meet publication standards and to improve interpretability.

Ethical and Data Source Clarification The manuscript should clearly state whether ethical approval was required for the use of hospital data and how patient confidentiality was ensured, even if the data are aggregated.

Minor Concerns

Several typographical errors and formatting inconsistencies are present throughout the manuscript.

Some sections are repetitive, particularly in the Introduction and Discussion.

Units and parameter descriptions in the appendix should be clarified and consistently presented.

The structure of some tables can be improved for better readability.

Suggestions for Improvement

Revise the entire manuscript for language quality and clarity.

Replace all missing citations and verify references.

Improve mathematical notation and provide clearer derivations.

Expand the methodology for parameter estimation and numerical implementation.

Provide full data and code access in line with PLOS policies.

Include quantitative validation metrics.

Revise figures and tables for better presentation.

Reframe the novelty claims in a more balanced manner.

Conclusion

The manuscript addresses an important topic and combines mathematical modeling with real data and optimal control analysis. With substantial revision focusing on language quality, citation completeness, data transparency, mathematical clarity, and methodological rigor, the study has the potential to make a meaningful contribution to tuberculosis control research. At present, major revisions are necessary before the manuscript can be considered for publication.

Reviewer #3: PLOS Global Public Health (PGPH-D-26-00093) Reviewer’s Comments The authors studied ‘Dynamical Behavior Analysis of 2-control Strategies on Tuberculosis Model. The authors must be commended for carrying out this research. As the paper is, technically correct formulation of the SEITR system, Standard application of Pontryagin’s Maximum Principle, Inclusion of sensitivity analysis and data fitting and clear policy motivation (treatment and distancing synergy). However, authors need to address the following suggestions to improve the manuscript: Comment 1: Abstract Please consider/incorporate the followings points in the abstract i. aim/ objectives ii. Methods applied iii. Results/ Findings iv. Recommendations to policymakers

Comment 2: Introduction Please consider/incorporate the following points in the introduction. i. Significance of the research ii. Please clearly indicate in a summarized form your observations about the related literature reviewed as compared to your research. Comment 3: Research gap and main contribution At the ending section of the introduction: i. Authors should clearly state the research gap in the manuscript. ii. SEIR/SEITR TB models with: treatment control, distancing or awareness control, PMP-based optimal control, sensitivity analysis have been extensively studied (including several papers already cited). Authors should compare their research with the existing literature and state what exactly is new in their research (main contribution) compared to existing studies. iii. The claimed novelty which comprises “composite analytical methodology integrating control-oriented stability decomposition with normalized forward sensitivity indexing” is not clearly demonstrated mathematically. Authors should check that.

Comment 4: Stability analysis The stability analysis must be improved. Authors analyzed only DFE stability. Authors should consider Endemic equilibrium (EE) existence, Local or global stability of EE and Backward bifurcation analysis. Since TB models, normally exhibit backward bifurcation and highly relevant. Comment 5: Sensitivity analysis Authors conducted so many sensitivity plots, with little insight. Parameter ranges are not clearly justified and some interpretations are inconsistent (e.g., γ showing weak PRCC but strong analytical sensitivity). Authors should correct that. Comment 6: Grammatical Errors There are some grammatical errors in the manuscript. For instance 1. “Pontryagain’s” → Pontryagin’s 2. Repetitive phrases (“It is noted that…”, “It is observed that…”) and 3. Overly long descriptive captions instead of analytical discussion

Comment 7: Inconsistencies in Notations 1. The authors used \lambda for both force of infection and adjoint derivative, 2. Parameters appear in equations but are not always defined immediately 3. Table 1 includes parameter \eta which does not appear in the model Authors should correct that.

Comment 9: In Text-Reference A lot of in-text references are missing in the manuscript. Authors should correct that.

Comment 10: Conclusion State the limitations and future work in this section. Comment 11: Cost-effectiveness analysis Cost-effectiveness analysis can be added to the control model to improve the manuscript. Remarks: Minor Revision Required Thank you

R1: Reviewer #2: The authors have adequately addressed all comments raised in the previous round of review. The revisions have significantly improved the clarity, methodological rigor, and overall presentation of the manuscript. The study presents a well-structured and technically sound analysis of the dynamical behavior of tuberculosis transmission under two control strategies. The modeling framework is appropriate, and the assumptions are clearly stated and justified. The statistical and mathematical analyses appear to be conducted rigorously, and the results are presented in a coherent and interpretable manner. Importantly, the conclusions are well supported by the data and align with the objectives of the study. The authors have also ensured compliance with data availability requirements, which enhances transparency and reproducibility. The manuscript is clearly written in standard English, with improved organization and readability compared to the previous version. Any minor typographical or grammatical issues noted earlier have been appropriately corrected. Overall, the manuscript now meets the publication criteria of PLOS Global Public Health and is suitable for publication.

Reviewer #3: Dear Editor PLOS Global Public Health(PGPH-D-26-00093R1) Reviewer’s Comments The authors studied ‘Dynamical Behavior Analysis of 2-control Strategies on Tuberculosis Model. I have checked the revised version of the manuscript according to the comments given. The authors have revised the manuscript according to the comments. I suggest that the manuscript can be accepted for publication PLOS Global Public Health after this minor suggestion. However, authors need to address the suggestion below to improve the manuscript: Comment on main Contribution The main contributions must not be stated as questions. Authors should correct that. Authors should be: 1. Specific: Focus on what is new (novel theory), the method, or data. 2. Employ Active Verbs: Begin them with phrases such as "We propose," "We discover," "We develop," or "We demonstrate" etc 3. The introduction should state the questions you asked, while the contribution section (or conclusion) should state the answers you found.

Remarks: Accept Thank you

Reviewer #4: Interesting study, here are my recommendations to improve the manuscript:

• Authors need to conform to international guidelines of scientific writing. Sentences like "doctors typically diagnose..." can be better written with "healthcare professionals diagnose..."
• The introduction section has information about the basics of tuberculosis and models design that are not necessarily important to the study. Can revise and shorten. See comment above too.
• In fact, it would be beneficial if you provide details about the current TB control strategy in Bangladesh and some background epidemiologic data about TB in Bangladesh (the country of interest) the introduction.
• tuberculin skin test is use to diagnose TB "infection"- clarify this to readers
• I assume that the first control strategy "treatment" involves treatment of active TB disease, not latent infection.
• Not sure if authors accounted for failure of TB treatment in the model?
• What is the prevalence of MDR-TB in Bangladesh and was this accounted for in the transmission model and cost-effectiveness model?

This is notable because many people tend to judge sources too quickly based solely on titles. The specific suggestions for checking abstracts, introductions, indexes, and bibliographies offer effective strategies for evaluating sources more thoroughly and efficiently.

This section reinforces an important point that is easy to overlook during research. Even a highly credible source is not very useful if it does not actually connect to the research question being studied.

The COVID-19 example was particularly effective because it demonstrates how source evaluation can change based on circumstances. By using a real-world scenario that everyone, especially in an online environment, has experienced, the discussion about currency becomes much more practical and relatable.

This introduction clarifies relevance in a straightforward manner. Research can sometimes feel complicated, so starting with a clear definition simplifies the evaluation process.

The examples in this section provide valuable insights into how the evaluation of sources can vary depending on context and purpose. By comparing casual information needs with those related to research or health, we can better understand the critical role that thoughtful analysis plays in assessing sources effectively.

This explanation is compelling because it emphasizes that many people mistakenly prioritize a source's trustworthiness over its relevance to the research question. The section clearly illustrates that assessing relevance should be the initial step in effectively evaluating information.

Evaluating sources can sometimes feel less straightforward than students expect; it does a good job showing how credibility is often determined by looking at multiple details together instead of relying on one factor alone.

The ethical and environmental concerns makes the conversation about AI feel more balanced instead of focusing only on convenience and productivity.

This section connects strongly to information literacy because it reinforces the importance of lateral reading and source evaluation. AI can be helpful for brainstorming or explanations, but this chapter does a good job reminding readers that it should not replace careful research.

A brief example showing what an AI hallucination might actually look like in a student research paper could make this concept even easier to recognize in practice. The citation example was useful because it shows how believable incorrect information can appear.

This explanation was especially helpful because many people assume AI-generated information is automatically correct if it sounds professional or confident. The discussion about plausibility versus accuracy does a good job explaining why verification and critical thinking are still necessary when using AI tools.

The Google Books section was useful because many people probably only think of Google as a search engine and not as a research support tool. Being able to preview books before requesting or purchasing them seems especially helpful for narrowing sources down more efficiently.

This practical tip makes the section easier to apply in real research situations. A lot of frustration during research comes from finding articles without full access, so this strategy could save students time with trouble shooting.

The citation tracing explanation was especially helpful because this is a feature many people probably overlook when using Google Scholar. Including examples like this makes the tool feel much more useful for finding current research and related studies.

Many students tend to use Google Scholar as their first resource without recognizing the importance of evaluating sources. This explanation effectively clarifies that while Google Scholar can be useful later in the research process, it should not be the primary starting point for research.

This explanation made interlibrary loan feel much simpler than it seemed initially. Before reading this section, the process appeared more complicated and difficult to access.

We shouldn't forget to support our local libraries so they can remain a part of the community, especially amid all the cutbacks to library resources. The reminder about public access helps show how community resources can support research and lifelong learning.

This is really awesome tool; Including a direct example or screenshot of what an interlibrary loan request form looks like could make this process easier to understand for first-time users who may feel intimidated by library systems.

Helpful in providing resources outside of their own school or town library.

This stood out because bias is often thought of as directly sharing opinions, but leaving out important information can also influence how people understand a topic or event. The comparison between different types of news sources was helpful in explaining how credibility and bias can vary across media platforms.

This section was helpful because it explains that sources are not automatically good or bad on their own. The usefulness of a source really depends on the purpose of the research project and the type of information being gathered. While completing research for this class I found myself running into this problem a couple of different times.

This part stood out because research projects can become overwhelming and frustrating when information starts piling up from different places; its not surpsing if slip up happen. The example template included in the chapter was helpful because it shows a realistic way to stay organized throughout the research process.

Organizing source types and information needs ahead of time seems like a good strategy for avoiding confusion and making the research process feel more manageable; making the process less stressful.

The section does a good job explaining that sources are not only used for citations, but also for developing ideas, understanding different viewpoints, and building stronger arguments.

Having multiple perspectives make the discussion more balanced and thoughtful. The emphasis on addressing multiple perspectives also connects well to critical thinking and stronger academic writing.

Puts this as a realistic approach to research by acknowledging that many students get frustrated with finding sources. Explaining sources as tools that support the entire research process helps make research feel more purposeful instead of just another assignment requirement.

The section does a good job combining definitions with practical guidance instead of only explaining the concept theoretically. Including a screenshot example from a database or library catalog could make the process even clearer and easier to recognize while searching for sources.

This comment is helpful- peer review involves much more than simply checking for mistakes. The section does a good job explaining how the process helps maintain quality and credibility within academic research.

The visual examples throughout this section make the difference between quantitative and qualitative data much easier to understand. Including images and real-world examples helps explain the concepts in a more practical way instead of only relying on definitions. It really helped me out I am a visual learner so having that was great!

helpful because typically associaiton in regards to data only with charts, spreadsheets, or statistics. The examples using videos, sound clips, and maps do a good job showing how information can be communicated in multiple formats depending on the situation or audience.

Super important to Note; The explanation here does a good job reminding readers that all sources still need to be evaluated carefully for credibility and bias, even when they come directly from someone involved in an event.

explains why understanding primary, secondary, and tertiary sources actually matters beyond just memorizing definitions. The connection to critical thinking and workplace skills makes the topic feel much more practical and relevant; outside of the classroom setting.

The Evolution of the Card Catalog System – Raymond Geddes<br /> accessed on 2026-05-13T21:39:37

This is a good point to make; explanation helps normalize revising and refining ideas throughout the research process.

This example stood out because it shows how research questions often explore larger social issues instead of looking for one simple answer. It also demonstrates how research questions can connect to community access, education, and civic engagement in meaningful ways.

This introduction was great because it connects research questions to everyday life instead of making them seem like something that only exists in academic settings. The examples throughout the section also make the difference between regular questions and research questions much easier to understand.

The 1960 Royal FP "special order"<br /> by [[Erik Bruchez]]<br /> accessed on 2026-05-13T21:18:30

This is important here:often people gather information quickly without thinking critically about it.

The emphasizes that research questions are not just another assignment requirement. A strong research question helps guide the entire direction of a project and makes the research process more focused and intentional.

Steve Job points out great key points and technology and how technology should be used and to be approached. Many company make there technology very complicated and when there is a issue with that technology where a user can not understand it the user may give up. What this video mentions is that technology should be simply and easy to understand so that all users can control it and overall have a great experience with that specific technology.

This is important (I feel like research paper get scary due to the wide variety of informaiton) because many students probably begin research with questions that are too broad or too simple to support an academic project. Including specific examples helps make the difference between the two much easier to understand.

The Royal KHM (1938)<br /> by [[Erik Bruchez]]<br /> accessed on 2026-05-13T21:06:46

My KHM holds this same distinction in my collection.

This section explains well that research topics usually become clearer over time rather than being fully developed at the beginning. That approach makes the brainstorming process feel more manageable and realistic for students who are still trying to narrow down their ideas.

The 5Ws structure makes mind mapping feel much more approachable and organized, especially for students who may struggle with where to begin brainstorming; which makes the whole process less stressful.

This section was helpful as it explains the purpose of a research plan in a simple and straightforward way.

Erik Bruchez describes how he used shellac and denatured alcohol to re-shellac his Yost 20 followed up with 600 grit sandpaper to smooth things back out as necessary.

The large promotional banner at the top of the Walmart page automatically rotates between images, which creates an accessibility issue. Module 2 explains that many users with motor impairments rely on assistive technologies like adaptive keyboards, head wands, or single-switch devices, and these tools cannot easily pause or control moving content. When the carousel changes on its own, it can move too quickly for screen readers to finish reading the text and makes navigation harder for users who cannot use a mouse. Because the movement happens without user input and may not offer clear pause controls, it fails the "Operable" principle by reducing user control over the interface.

Each product tile on the Walmart page uses proper HTML structure for elements like product names, prices, and "+Add" buttons. Module 2 explains that content must be readable by a wide range of assistive technologies, including screen readers, braille displays, and screen magnifiers. Because these elements are coded in a way that tools like NVDA, ChromeVox, and braille readers can interpret, users with visual or motor impairments can reliably access the same information. This supports the "Robust" principle by ensuring the page remains usable across different devices, browsers, and assistive technologies as they evolve.

The search bar at the top of the Walmart page can be accessed and used with only a keyboard, without requiring mouse-based actions like hovering. Module 2 explains that not all users can operate a mouse, especially those with motor impairments, so interactive elements must be useable through alternative input methods. Because the search bar supports keyboard navigation, it follows the "Operable" principle by allowing users to interact with the interface in multiple ways.

This Walmart section shows many promotional tiles at once, each with different colours, images, and text styles. As Module 2, explains users with dyslexia, ADHD, or autism can struggle with cluttered or visually busy designs because they increase cognitive load. Since the layout is not simple or predictable, it violates the "Understandable" principle by making the content harder to process.

This brings up a good point; terminology during research is very important. Without learning the language commonly used within a topic, it would be easy to miss valuable sources or information while searching databases and other academic resources.

This stood out because its comon thougt process to assume jumpint into scholarly journal articles when starting research. Beginning with background reading first seems much more realistic and ideal since it helps build a foundation and understanding of important terminology before moving into more advanced academic sources.

Not immediately having a perfect research question makes the research process feel more realistic. A lot of students probably assume they are supposed to know exactly what they want to research right away, when in reality good research often comes from exploring different ideas, asking questions, and slowly narrowing things down through curiosity and critical thinking. Picking on topic can be very challenging.

Starting off with this sentence is very important to many users its a strong topic to talk about and when i read the whole article this stood out to me the most. This sentence says everything about Dark patterns they lead an individual to making a decision that the user does not know what they are doing and that they don't understand it completely. For example when you go and sign up for any entertainment subscription there may be hidden fees that the user does not notice until they pay everything, when that happens the user does not trust that company anymore due to the unprofessional experience.

The research plan section stood out because it makes the research process feel more organized and intentional instead of overwhelming. Breaking research down into categories like source types, databases, and information needed seems like an effective way to avoid wasting time and create a clearer direction before starting a major project

The explanation of annotations being more than just summaries was helpful because it highlights how research is supposed to be an active process instead of just collecting sources.

via Tyler Alan Macek at https://www.facebook.com/groups/705152958470148/posts/1255908216727950/

A black light is potentially better, but a bright light should work too.

via Leighton Jeffy at https://www.facebook.com/groups/705152958470148/posts/1255908216727950/

Sounds roughly similar to

via Luke Fuji at https://www.facebook.com/groups/705152958470148/posts/1255908216727950/

https://discord.com/channels/639936208734126107/639938269030907914/1302694827682697330

Pelicram's Shellac Rescue Formula aka The Cowboy's Delight.

This will help bring back a deeper black color shellaced panels which have been yellowed and damaged by UV over the years. With enough elbow grease it will remove the old shellac completely but it takes a very long time and you're likely to damage any decals present on the panel. In most cases the procedure described below will be sufficient to restore the appearance to an acceptable level.

The recipe: - 70% Light machine oil. - 30% IPA (Isopropyl Alcohol) or White/Mineral Spirits.

Ideally use an oil that is dissolved into the IPA/Mineral Spirits, if they settle into separate layers make sure you shake the mixture thoroughly before applying.

Mix the oil and solvent in something like a dropper bottle or similar vessel for convenient application.

• Clean part with Fulgentin (Or general purpose cleaner of your choice) and wipe dry.
• Apply oil/ipa mix to part and rub in lightly with clean microfiber cloth or shop towel. Use plenty of the mix, it should not feel dry.
• Wipe with microfiber cloth after 15 minutes to get rid of any excess.
• Do not apply any kind of wax (like Renessaince Wax) afterwards, from my testing it will bring back the haziness.

evaluating information stood out to me because misinformation is so common now, especially online and on social media. I think this part is very relatable to everyday life because people constantly have to decide what information is trustworthy and what is misleading. In professional settings, especially in human services, evaluating information carefully is important because decisions can directly impact other people and the support or resources they receive

I enjoyed that this example shows information literacy as something we use in everyday life and not just for school assignments. There is so much information online about dieting and health trends now that people really have to evaluate what sources are trustworthy and what actually fits their own health needs. My brother has diabetes and fatty liver disease, and I have seen firsthand how important it is to find reliable information instead of just following random advice or trends online.

equalising everyone on the same level

This is foreshadowing Tea Cake possibly dying. The dog may have been rabid. Back then they didn't know what that was though

Janie hadnt felt jealous with anyone else she has been with except for Tea Cake. Her past relationships may have caused a toll on her jealousy control too

He was insecure about him being older than Janie and not much good looking as Janie. He knew Janie was too good for him and he was only getting older.

This horizontal scrolling section featuring products like coffee, protein shakes and tea almost certainly has no meaningful alt text on any of the product images, meaning a screen reader user would have absolutely no idea what they are looking at, which directly violates the Perceivable principle from POUR. The horizontal scrolling format also raises concerns around the Operable principle since keyboard-only users may find it difficult or impossible to navigate through all the products. Simply adding descriptive alt text like "Kicking Horse Decaf whole bean coffee" to each image would make a significant difference for visually impaired users.

This section shows a row of product images including dumbbells, resistance bands, a water bottle and fitness trackers, all of which are almost certainly missing meaningful alt text descriptions. A screen reader user would have no way of knowing what any of these products actually are, which is a direct violation of the Perceivable principle from POUR. It is also worth noting that having so many similar looking product images lined up in a row with no clear labels makes this section visually overwhelming, which could be an issue for users with cognitive disabilities too. A simple fix would be adding descriptive alt text to every product image like "purple 3lb dumbbells" or "Gritin resistance bands set of 5."

The large image of the red handbag with strawberries in the background is a really nice visual but almost certainly has no descriptive alt text, which means a screen reader user would completely miss what is being advertised here. This is a violation of the Perceivable principle from POUR since the information is essentially invisible to anyone using assistive technology. The "Shop all" link underneath also suffers from the same vague labelling issue mentioned in other sections, where a screen reader user would have no context about where that link actually leads.

The images in this section like the yellow dress and striped shorts are clearly being used as navigation links to different fashion categories but likely have no descriptive alt text attached to them. This violates the Perceivable principle of POUR since a screen reader user would get no useful information about what each image actually shows. The subcategory labels like "Women," "Men," "Premium Brands," and "All fashion" are at least somewhat helpful, but without proper alt text on the images the section is still pretty inaccessible for visually impaired users.

The images in this section like the tote bag, hat and soccer ball are being used as clickable links but almost certainly have no meaningful alt text, meaning a screen reader user would have no idea what they are clicking on. This is a pretty clear violation of the Perceivable principle from POUR that we covered in Module 2. On top of that labels like "Outdoorsy dad" and "Cool dad" are fun visually but are vague and do not really tell you what products are actually in each category, which goes against the Understandable principle too.

The yellow button actually does a good job with colour contrast and the label "Sign in securely" is clear and specific, which aligns with the Perceivable and Understandable principles from POUR. However the heading above it, "Sign in for your best experience," reads more like a marketing slogan than a functional description, which would not be very helpful for someone navigating by headings on a screen reader. Something simpler like "Sign in to your account" would honestly communicate the same thing in a much more accessible way.

Another term related to the internet performance is latency, so we just talked about bandwidth, which is the throughput or the speed that you can upload and download information latency, then, is a measure of the time. It takes a piece of data to reach its destination. So, it's typically measured in milliseconds or thousandths of a second. A ping activity, which will measure the latency of your network. So, ping is a networking utility used by Network administrators to measure the administrators to measure the latency on the internet. So, what you can do is, you can go to a website and use their ping utility to test the reachability of certain hosts.

大多数人认为小企业面临资源限制是永恒的约束。但作者引用CEO的话表明，AI正在重新定义这些约束，认为曾经被视为限制的因素现在已不再是真正的障碍，这挑战了关于小企业资源限制的传统观念。

大多数人认为AI工具的采用障碍主要是成本问题或技术复杂性。但作者指出，真正的障碍在于现有工具和培训未能适应小企业的运营方式，导致AI使用仅停留在基础聊天层面，这挑战了关于AI采用障碍的主流认知。

大多数人认为AI技术会扩大大企业和小企业之间的差距，因为大企业有更多资源采用新技术。但作者认为AI是首个能够缩小这种差距的技术，因为它能以相对较低的成本提供强大的能力，使小企业能够获得与大企业相当的工具和效率。

大多数人认为数据库优化需要人类数据库专家的经验和知识，但作者认为AI可以独立发现并改进核心数据库算法。这挑战了数据库工程领域的传统实践，暗示AI可能在最基础的系统组件上实现超越人类专家的优化。

大多数人认为数学证明需要人类直觉和创造力，但作者认为AI工具可以显著加速数学发现过程，甚至帮助人类找到更严谨的证明。这挑战了数学研究作为纯粹人类智力活动的传统观念，暗示AI可能成为数学家的真正合作伙伴而非简单工具。

大多数人认为AI系统的硬件设计需要人类专家精心设计，但作者认为AI本身可以设计出比人类更高效的硬件电路。这挑战了传统硬件工程领域的共识，暗示AI可能在最底层的硬件设计上超越人类专家的直觉和经验。

大多数人认为AI实验室之间的竞争是为了技术进步和社会福祉。但作者暗示这种竞争更像是为了赢得价值数十万亿美元的自动化劳动力市场，这种'赢家通吃'的动态进一步加剧了顶级研究者的薪酬差距，可能带来极小的社会收益。

大多数人认为随着AI普及，薪酬差距可能会缩小或趋于稳定。但作者认为，随着AI用户数量和使用频率的增加，'超级明星效应'只会变得更加重要，顶级AI研究者的薪酬差距可能会进一步扩大，甚至出现1亿美元的年薪也不够的情况。

大多数人认为AI智能爆炸的速度取决于模拟顶尖研究者与普通研究者能力的巨大差异。但作者认为，如果薪酬差距主要是由'超级明星效应'而非真实能力差异驱动，那么研究者之间的实际能力差距可能小得多，这对AI发展速度的预测有重要影响。

大多数人认为只有那些真正卓越的'10倍研究者'才值得超高薪酬。但作者认为，即使是只有2倍能力的AI研究者，由于其工作可以影响数十亿用户，微小的质量优势也能产生巨大价值差异，从而获得远超中位数的薪酬。

大多数人认为顶级AI研究者获得超高薪酬是因为他们能力远超常人，可能是10倍甚至100倍更优秀。但作者认为，即使超级明星研究者只比普通博士后好一点点，薪酬差距也会非常大，因为'超级明星效应'会将微小的能力差异转化为巨大的薪酬差异。

A dependent variable: strength of LOT and ASSN inputs on the same pyramidal cells

The results of the study support the notion that there is a critical period for smell input. The results support the authors' hypothesis

Something to be researched further

Evidence of a critical period

insight from past study

This is a very exciting finding because it strongly suggests that there is a critical period for smell

more background on prior studies

What the study is examining - hypothesis

Results of the study

These citations seem ggenerated by zotero and don't seem to follow MLA style

why all caps?

This claim is somewhat belied by all the quotations from other scholars, who are acknowledging that Loy's portrait poems say something about her own art and life.

This paragraph feels like you're struggling a bit to differentiate your argument from the critics. You've got a latent argument here that you could make, in the idea that the boundaries between self and other are permeable, particularly in the Paris community of expatriate artists. By returning to the setting of Paris, you could argue that Loy found herself by portraying others in Paris, a place where artists lived and worked in close proximity, inspiring one another with their bold experiments in both art and life.

Fascinating insight. Does this suggest that the boundaries of selfhood are permeable? Or that all innovative artists share certain qualities and characteristics that distinguish them from the average joes? Or perhaps, that Loy uses portraiture as a mode of introspection, looking outward in order to better understand herself.

Good idea to put the portraits side by side, though I think it calls to mind some of her later photos by Man Ray more!

Did I say that?! Where? (include parenthetical)

Good connection to Feminist Manifesto, but the text is too tiny to read. Better to quote only a passage.

Again, we may not need this detail, nor the "ultimately"

Do we need these details, or can you just say:

Lewis argues that Loy's abstraction wasn't abstract...

Good point, but this close reading lacks the specificity of the previous one, so it seems like another example of the same thing, rather than an example that generates a new, different insight or illuminates another dimension of her portraiture.

Personally, I like your writing style better than his. Maybe you could make the font smaller? It's like he's yelling over you!

Cool idea -- what tactics? And how does she make use of readymades in this poem, a tactic typically associated with Marcel Duchamp rather than Joyce?

unnecessary verbiage that doesn't really tell us any more about his argument or the poem -- just cut it.

Wow! How did you animate the poem image with the boldface? Amazing!

This is really good. I think the easiest way to clear up confusion is to say something like: Most of the photographic and literary portraits of Mina Loy made by other people were created during her Paris years. Loy also created many portraits during her Paris years: of the ten poems she published, 5 are portrait poems. It is these 5 portrait poems that I will examine here.

So you're talking about portraits of Loy made by other people?

What do the portraits have to do with the Paris poems. I want to hear about both, but this seems like a jarring shift, after you just announced a purpose to look at the Paris poems. Perhaps you could smooth the transition by emphasizing in the previous paragraph and this one that Loy's Paris years were distinguished by a market interest in portraiture. Though Loy made portraits throughout her career, she did so with a particular intensity in both poetry and prose in the Paris years. (if this is true, that is!)

Also, it would be helpful to know whether you're talking about portraits of her or by her or both.

The Bowery years were important artistically and poetically, so should probably have their own frame in this timeline.

Maybe this level of detail would be better in a narrative than a timeline?

Avoid equating the poem with autobiography. You might say Haweis was having an affair, a detail Loy alludes to in her poem about childbirth, "Parturition."

Why Senior Developers Fail to Communicate Their Expertise

• Conflict of Interest: Senior developers and business leaders operate in different "loops." The business prioritizes the Speed Loop (reducing uncertainty by getting to market fast), while senior developers prioritize the Stability Loop (managing complexity to ensure the system remains debuggable and reliable).
• Vocabulary Mismatch: Communication fails because developers talk about "complexity" and "technical debt," which sound like excuses to non-technical stakeholders. Stakeholders are focused on "uncertainty" and "growth."
• The "Solution" Frame: To communicate effectively, seniors must frame their concerns as solutions to the business's problems. Instead of saying "no" to complexity, they should offer "something quicker" (reusing existing tools, Google forms, or minimal UI changes) to help the business learn faster without bloating the code.
• AI as a Destabilizer: AI-generated code accelerates the Speed Loop but creates a "complexity explosion" that threatens long-term stability. The senior developer’s role is shifting toward that of an Editor, responsible for extracting stable, scalable logic from the rapid "vomit drafts" produced by AI and junior staff.
• The Proposed Split: A potential workflow involves maintaining two versions of a system: a "Speed" version for rapid experimentation and a "Scale" version that is curated, stabilized, and owned by senior architects.

Hacker News Discussion

• The "World Model" Gap: A top-voted comment argues that true expertise is an internal "world model" or intuition that is inherently difficult to put into words. It isn't just a list of facts but a deeply integrated understanding of how systems behave.
• Recipe vs. Physics: Commenters distinguish between "recipe-following" developers (who use tools without understanding the underlying logic) and "physics-based" developers who understand the fundamental nature of computation.
• The Role of Failure: Discussion emphasized that senior intuition is built primarily through experiencing failures and reflecting on them over many years. This mental shift often happens when a developer stops trying to learn syntax and starts focusing on solving specific visions or problems.
• The "Senior" Title: There was significant debate about whether "senior" is a measure of years (tenure) or actual merit, with many noting that some developers with decades of experience still only follow recipes and lack the "world model" required for true expertise.
• Abstraction Struggles: Some users noted that while they have a "physics-level" understanding of physical sciences (like chemistry or biology), software abstractions feel arbitrary and "anti-physicalist," making it harder for some brilliant minds to build that same level of intuition.

Summary of "Googlebook" Discussion (Hacker News)

• The Concept: Googlebook is presented as a high-end hardware play—a "premium upgrade" to the Chromebook—intended to shift the user experience from traditional apps to an LLM-native environment powered by Gemini.
• The Vision: Some users see this as a "moonshot" to make apps irrelevant, replacing them with raw data feeds and AI-driven visualizations, potentially creating a "sci-fi" style user interface.
• Execution Skepticism: A dominant theme is doubt regarding Google's ability to execute. Commenters cited current frustrations with Gemini (cutting off mid-sentence, poor Maps integration, and hallucinations in Sheets) as evidence that the tech isn't ready for a primary OS role.
• Target Audience: Discussion suggests Google is targeting "Chromebook natives"—students who grew up in the Google ecosystem and are now entering university or the workforce and want a "pro" version of their familiar tools.
• Public Sentiment on AI: There is a sharp divide in perceived AI adoption. Some argue "normies" (non-tech users) love AI for daily tasks and creative fun, while others claim people increasingly resent AI being "forced" into every product.
• Competitive Landscape: Comparison was drawn to historical shifts (like the move to Mosaic/Web browsers). While some see this as an inevitable evolution, others believe Apple’s approach—gradually improving Siri and app intents—is more practical than trying to eliminate apps entirely.

What is unique about the study is that it is looking at a change and not just a mechanism for likes and dislikes taste-wise

Interesting that change in natural aversion to bitterness could be because the taste cannot be sensed as well

Dopamine is associated with pleasure and motivation, so its connection to taste pathways fits into the system

Uncertainty in unexpected results between MSG and MPG solutions, possible interactions between sodium and umami signals

Results from study support hypothesis and previous research on the modification of taste preference through experience

Cellular populations respond to a mix of negative and positive tastants with some bias in tendency

As expected, the largest populations of Prkcd-positive neurons responded to the bitter

Hypothesis for neural activity mechanisms in connection to taste quality response

In addition to behavioral changes, study is interested in neuronal activity associated with them

1 - this website offers a variety of subheadings and embedded links that help the user navigate the website and relevant topics in Toronto currently such as the cherry trees blooming in high volume areas of the city, namely High Park. However, the what's new page does not include the list of long standing or yearly events which the city hosts throughout the year, such as Nuit Blanche.

2 - The subheadings on the site are very simple and easily understood, which would be beneficial for Toronto's diverse communities and individuals who may have a difficult time navigating a website with a more complex or word heavy interface.

3 - The homepage of the website only showcases a very small amount of information one using the website may access, with only the search bar to find what one needs, which may be difficult for people who may not know their exact search query or have difficulties with searching.

4 - The page does offer it's own translation feature, which would be expected to be more reliable than the Google offered automatic feature as one would assume the city would employ real translators to translate the page. This feature would be beneficial for Toronto's healthy population of non-english or ESL individuals.

5 - The phone number 311 is not specified as being a phone number for the city, which may make it confusing or difficult for a new resident to contact the city for relevant services or assistance.

We need to add filtering options.

In the table I would like to see Disease, Clinical trial number + Title, Geography (a specific country or international), Phase, Status, Enrollment, Primary Completion Date.

I would like to add additional sections: 1. Sales (wee what we have in the current US) 2. Clinical Evidence 3. Technology Features - we are collecting additional features for certain technologies. These features are technology dependent.

I think you might be able to make this argument more effectively if you emphasized her resistance to the sexualizing male gaze rather than to patriarchal pressures more broadly. Your wonderful analyses of the paintings show HOW they resist the male gaze. It's much harder to show how she's resisting something as abstract and general as patriarchal pressures.

What does "this" refer to? What are the patriarchal assumptions she's resisting?

Ahh, this is exactly the kind of comparison that might work better above in the place of a generalization about male painters, but tell us about the stylistic differences. Is Degas an example of patriarchal attitudes? or of the modernist erotic destruction of the female body?