Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
DESCRIBED WELL ENOUGH, 1:1 for the deposited proteomics. PRIMARY RESULT REPRODUCED EXACTLY: Suppl. Fig. S1f FASN acetyl-Lys site ratios (R2177C/WT) recomputed from the deposited Proteome Discoverer Quan .msf (PXD045908) match all 7 quantifiable sites at full float precision (K670 5.74, K776 2.61, K1582 2.5, K1704 0.6443781, K1927 0.743032027, K2206 3.828036828, K2436 1.146144002); K2449 WT non-detection confirmed. The earlier K2206 discrepancy was RESOLVED by using RT-anchored Minora-style Match-Between-Runs (the peptide elutes as two peaks in R2177C; the paper quantifies the WT-aligned peak -> 9088281/2374136 = 3.8281, exactly the printed value). Compute ran on «our HPC» SLURM «job» (node n094). FIG 1E (ubiquitin volcano) PARTIAL/UNCHECKABLE: the 12.2 GB ubiquitin search DB was downloaded and parsed - FASN shows 69 GlyGly(K) sites across all 32 runs with high abundance (qualitatively consistent with FASN ubiquitinylation), but the run->condition design needed for the WT-vs-R2177C+MG132 n=4 two-way-ANOVA volcano is NOT in the deposit (empty PD factors, no SDRF, no Fig 1e source data), so the specific volcano cannot be reproduced 1:1. NOT ATTEMPTED (out of scope, no deposited pipeline): lipidomics, 14C radio-tracing, siRNA/Western blots, mouse phenotyping, the human FASCINATE-1/TVB-2640 trial (restricted), and the patient exome (clinical, not deposited). FABRICATION FLAG (minor): the source-data sheet S1f prints R2177C='5.74' for K2449, identical to K670 and inconsistent with K2449 being WT-n.d. - likely a transcription artifact; flagged for human review, not a fabrication of the underlying data (which reproduces exactly).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 94assessed: 2026-06-19 ⛓ 273bb8d1ec78
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-19
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-20no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether the level of endogenous fatty acid synthesis (de novo lipogenesis, driven by FASN) determines how dietary polyunsaturated fatty acids (PUFA) are taken up and incorporated into complex lipids (triacylglycerols) in humans and mice.
- ★ FASN activity critically determines the use/incorporation of dietary PUFA into complex lipids in mice and humans finding
- ★ Combining FASN inhibition with PUFA supplementation decreases liver triacylglycerols in high-fat-diet-fed mice finding
- ★ FASN inhibition increases PUFA uptake via the lysophosphatidylcholine transporter MFSD2A mechanism
- ★ PUFA incorporation into TAG after FASN inhibition is DGAT2-dependent mechanism
- ★ The de novo FASN Arg2177Cys variant causes hyperacetylation and hyperubiquitinylation of FASN, leading to reduced protein stability and hypofunction mechanism
- ★ Pharmacological FASN inhibition (TVB-2640) in NASH patients lowers SAFA and raises PUFA in plasma TAG composition finding
- Overexpression of the deacetylase HDAC3 reduces FASN R2177C acetylation, subsequent ubiquitinylation, and stabilizes the protein finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Lipidomics (plasma TAG fatty acid composition, lipogenic index) | index patient (FASN Arg2177Cys) vs. father/adult/child controls | none (endogenous de novo FASN variant) | plasma TAG fatty acid class composition, SAFA/MUFA/PUFA, lipogenic index | — |
| Lipidomics (plasma TAG fatty acid composition) | NASH patients, FASCINATE-1 clinical trial | drug (50 mg FASN inhibitor TVB-2640) vs placebo, 12 weeks | SAFA, PUFA (18:2, 20:5, 22:6) composition in plasma TAG | — |
| 14C-acetate incorporation into lipid fraction (FASN activity assay) | patient-derived fibroblasts vs. age/sex-matched control fibroblasts | endogenous FASN variant (Arg2177Cys) | FASN enzymatic activity | scintillation counting |
| Cycloheximide chase / Western blot, co-IP | HEK293T cells overexpressing HA-tagged WT or R2177C FASN | overexpression of WT vs. R2177C FASN, ± MG132, ± HDAC3 | protein stability, ubiquitinylation, acetylation, phosphorylation of FASN | Western blot |
| Mass spectrometry (proteomics) of immunoprecipitated FASN | HEK293T cells overexpressing HA-tagged WT or R2177C FASN | MG132 treatment, FASN variant | ubiquitinylated and acetylated peptide/site abundance | mass spectrometry |
| Radiolabeled fatty acid uptake and oxidation assay (14C-oleate, 14C-DHA) | HuH7 hepatoma cells | FASN inhibitor TVB-2640 (500 nM) vs. DMSO | cellular fatty acid uptake and oxidation | scintillation counting |
| Stable isotope tracing (deuterated palmitate, oleate, DHA) | HuH7 cells, secreted lipoproteins and cell pellets | FASN inhibition (TVB-2640) ± etomoxir | incorporation of labeled fatty acids into secreted/cellular TAG species | mass spectrometry-based lipidomics |
| siRNA knockdown screen of TAG-synthesis enzymes (DGAT2, AGPAT1, AGPAT2, DGAT1, LPIN1, GPAM) and MFSD2A | HuH7 cells | gene knockdown ± FASN inhibitor TVB-2640 | TAG PUFA/SAFA composition, DHA uptake and secretion | — |
- ▼ Lipogenic index (16:1/18:2 ratio) was reduced by half in the FASN-variant patient compared to controls ~2-fold reduction
- ▲ Highly unsaturated plasma TAG species, especially DHA (22:6), were strongly increased in the patient relative to controls, while smaller/low-saturation species were decreased
- – NASH patients treated with TVB-2640 for 12 weeks had lower SAFA and higher PUFA in plasma TAG composition than placebo P<0.0001 for both SAFA and PUFA
- ▲ TVB-2640 treatment increased DHA uptake in HuH7 cells
- ▼ DGAT2 knockdown (but not AGPAT1/2, DGAT1, LPIN1, or GPAM) reduced the higher TAG PUFA composition induced by FASN inhibition
- ▼ MFSD2A knockdown diminished DHA uptake/secretion and abrogated the PUFA-raising effect of TVB-2640 on TAG composition
- ▲ Upon FASN inhibition, deuterated DHA incorporation into secreted TAG was higher than deuterated oleate or palmitate, while palmitate incorporation decreased and oleate was unaltered
- – FASN R2177C showed decreased protein stability and increased ubiquitinylation and acetylation compared to WT FASN
- pvalue P=0.0022 (FASN activity (14C-acetate incorporation) in index patient fibroblasts vs. controls)
- pvalue P=0.0094 (Western blot quantification of FASN protein stability (WT vs R2177C) after cycloheximide chase)
- pvalue P<0.0001 (SAFA in plasma TAG, TVB-2640 vs placebo NASH patients (n=25 placebo, n=28 TVB-2640))
- pvalue P<0.0001 (PUFA in plasma TAG, TVB-2640 vs placebo NASH patients (n=25 placebo, n=28 TVB-2640))
- pvalue P<0.0001 (Change in 18:2 (omega-6) TAG residues, TVB-2640 vs baseline, paired t-test)
- pvalue P=0.0350 (Change in 20:5 (omega-3, EPA) TAG residues, TVB-2640 vs baseline, paired t-test)
- count n=4 (Ubiquitinylated FASN peptide abundance comparison, R2177C + HDAC3 vs R2177C + empty vector)
- count adult controls n=9, child controls n=6 (Lipogenic index and plasma TAG composition control groups for index patient comparison)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study combines a case report (a pediatric patient with a de novo FASN missense variant), cell-based/biochemical mechanistic experiments (patient fibroblasts, HEK293T overexpression, HuH7 knockdown/tracer studies), and analysis of a human clinical trial (FASCINATE-1, FASN inhibitor TVB-2640 vs. placebo in NASH patients) alongside mouse lipidomic comparisons. Group comparisons were mainly analyzed with unpaired or paired two-tailed Student's t-tests, and one two-way ANOVA was used to identify differentially abundant ubiquitinylated peptides in a proteomics dataset. Results are reported as mean ± SEM with per-figure sample sizes and exact or thresholded p-values, without an explicitly stated multiple-comparisons correction or software package for statistics.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| unpaired two-tailed Student's t-test | Fig. 1a, FASN activity (14C-acetate incorporation) in patient vs. control fibroblasts | n = 6 | not stated |
| unpaired two-tailed Student's t-test | Fig. 1c, western blot quantification of FASN protein turnover (WT vs. R2177C) | n = 3 | not stated |
| two-way ANOVA | Fig. 1e, volcano plot of differentially abundant ubiquitinylated FASN peptides (WT vs. R2177C) | n = 4 | not stated |
| unpaired two-tailed Student's t-test | Fig. 2c, plasma TAG fatty-acid class composition, NASH patients on placebo vs. TVB-2640 | n = 25 (placebo), n = 28 (TVB-2640) | not stated |
| paired two-tailed Student's t-test | Fig. 2d, within-group change in omega-6/omega-3 TAG residues from baseline to 12 weeks (placebo or TVB-2640) | n = 25 (placebo), n = 28 (TVB-2640) | not stated |
| unpaired two-tailed Student's t-test | Fig. 3a, DHA/oleate uptake in HuH7 cells, mock vs. TVB-2640 | n = 6 | not stated |
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Multiple separate unpaired/paired t-tests are used across many individual figure panels and comparisons (e.g., Fig. 1a, 1c, 2c, 2d, 3a) without a stated correction for running many tests.↳ Could also: A single ANOVA (or mixed-effects model) with a post-hoc correction such as Tukey HSD, or a Benjamini-Hochberg FDR adjustment applied across the family of comparisons — This would control the family-wise error rate or false discovery rate when many pairwise comparisons are drawn from related experiments, which is a standard consideration when the same or related samples are compared repeatedly.
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The proteomics volcano plot (Fig. 1e) identifies differentially abundant ubiquitinylated peptides using a two-way ANOVA with a P < 0.05 and fold-change > 1.5 threshold.↳ Could also: Applying a Benjamini-Hochberg (or similar) FDR correction across the peptide-level comparisons — Proteomic/peptide-level datasets typically involve many simultaneous statistical tests, and an FDR-based q-value cutoff is a widely used way to account for that multiplicity while retaining sensitivity.
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Variability is summarized as mean ± SEM throughout the figures, including for small groups (e.g., n = 3–6).↳ Could also: Reporting standard deviation (SD) or a 95% confidence interval alongside or instead of SEM — SD directly conveys the spread of the raw data, and a CI conveys the precision of the estimated effect; both are often considered informative complements to SEM, particularly for small sample sizes where SEM can visually understate variability.
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Comparisons with small sample sizes (e.g., n = 3–6 in several cell-based assays) were analyzed with Student's t-tests, which assume approximately normal data.↳ Could also: A non-parametric alternative such as the Mann-Whitney U test — Non-parametric tests do not rely on a normality assumption, which can be a useful alternative when sample sizes are small and normality is difficult to assess.
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In the human trial data (Fig. 2c, 2d), treatment-vs-placebo group differences and within-group baseline-to-12-week changes are each assessed with separate t-tests.↳ Could also: An ANCOVA or mixed-effects model that includes baseline value as a covariate and treatment as a fixed effect — Such a model can jointly account for both between-group and within-subject (baseline) variation in a single framework and can increase statistical power by controlling for baseline levels rather than analyzing between- and within-group comparisons separately.
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The gene-knockdown screen (Fig. 3e–h, e.g., DGAT2, AGPAT1/2, DGAT1, LPIN1, GPAM, MFSD2A) compares each knockdown condition against a common control.↳ Could also: A one-way ANOVA across all knockdown conditions with a post-hoc test such as Dunnett's test (designed for multiple-treatments-vs-one-control comparisons) — Dunnett's test is tailored to the many-groups-vs-one-control design and controls the family-wise error rate across the simultaneous comparisons more directly than a series of independent t-tests.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 38167725
Title: Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use Journal: Nat Commun 15:45 (2024) · DOI 10.1038/s41467-023-44364-y · PMCID PMC10762034 Authors: Worthmann, Ridder, Piel, Evangelakos, … (Heeren lab, UKE Hamburg + Sagimet)
What kind of paper
Primarily a wet-lab metabolism study: lipidomics, radioactive FA tracing (¹⁴C-oleate/¹⁴C-DHA), siRNA knockdowns, Western blots, mouse HFD feeding, a human NASH trial (FASCINATE-1 / TVB-2640, NCT03938246), and clinical exome sequencing of one FASN-variant patient. Almost none of this is a reproducible computational pipeline.
In scope (pipeline-derived, public data)
The ONLY publicly deposited computational dataset is the mass-spec proteomics:
- PXD045908 (PRIDE/ProteomeXchange). Affinity-purified (IP) FASN from cells expressing WT vs the patient variant R2177C-FASN, analysed for ubiquitinylation and acetylation site occupancy.
- The deposit ships both the Thermo
.rawfiles AND the authors' Proteome Discoverer search results as.msfSQLite databases — so the reported figures can be regenerated directly from the deposited search output, and independently re-searched from the raw files (P16 third-party tool).
| Result | Figure | Pipeline | Data | Status |
|---|---|---|---|---|
| FASN acetyl-Lys site ratios R2177C/WT | Suppl. Fig S1f | Proteome Discoverer 2.4/3.0 + Sequest HT + Minora (label-free) | 231004_BS_CS1_CS2_Quan_3.msf (2 raw: WT, R2177C) |
reproduced (6/8 sites exact) |
| FASN hyper-ubiquitinylation (volcano) | Fig 1e | Proteome Discoverer + Sequest HT + Minora, GlyGly(K), two-way ANOVA, n=4 | 220328_HVSleinUball.msf (32 raw) |
in progress |
Pinned search parameters (Methods + PRIDE)
Trypsin, ≤2 missed cleavages; fixed Carbamidomethyl(C); variable Oxidation(M), pyro-Glu(Q), Acetyl(protein N-term + K); for the Ub search GlyGly on K. DB = human SwissProt (Dec-2021, 20,365 entries). FDR < 0.01 (Percolator). Quant = Minora, log2, median-normalized. Fig 1e: n=4, two-way ANOVA, highlight P<0.05 & |FC|>1.5.
Out of scope (not attempted — not a public computational pipeline)
- Lipidomics (Lipidyzer/SCIEX) — proprietary platform, source numbers only.
- ¹⁴C radio-tracing, siRNA, Western blots, mouse phenotyping — wet-lab.
- Human FASCINATE-1 trial data — restricted, available only under MTA via
Sagimet (
«email», up to 3 yr) → drop classdata_restricted. - Patient exome (FASN R2177C discovery; BWA/SAMtools/Pindel) — clinical, not
deposited →
no_data_accession/data_restricted.
Reproduction strategy
- Verify-from-deposit (primary): parse the
.msfSQLite (PD result DB), extract FASN modified-peptide abundances per run, recompute site ratios (S1f) and the differential-abundance volcano (Fig 1e). - Independent re-search (P16, secondary): convert
.raw→mzML and run MSFragger/FragPipe with the same mods to confirm the FASN modification sites.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Strong reproduction of the deposited proteomics: all 7 quantifiable Suppl. Fig. S1f FASN acetyl-Lys R2177C/WT ratios were recomputed from the public PXD045908 .msf and match the reported values to full float precision, and K2449 WT non-detection is confirmed — deviations are rounding only. The only deficiencies are on the data-availability/authors' side: Fig 1e's ubiquitin volcano is uncheckable because the deposit lacks the run→condition design key (empty PD factors, no SDRF), and the source-data sheet contains a flagged K2449 '5.74' transcription artifact. Per the rubric these deposit gaps are q1/q2/q3 issues, not a derivability defect, so q5/q7 stay green while overall quality is solid with explainable deviations (yellow).
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.