Live·Open questions in longevity research

Supplementary materials to the preprint on the hypothesis benchmark

The main text is the preprint. Appendix lettering continues from the main text, where Appendix A gives an example of five hypotheses. Table S lists all selected hypotheses with their reference findings and the judges’ scores.

Appendix B. Prompts given to the predictors§

The substantive prompts used in the experiments are reproduced below in their original English; technical instructions on the answer format and the absence of tools, appended to some requests, are preserved in the published request records. Bracketed placeholders were replaced with the full text of the task and the hypotheses. The full request records for each model are in the published materials listed under “Data and code availability”.

B.1. The detailed prompt

System prompt:

You advise researchers which causal hypotheses deserve experimental testing in the biology of ageing.
Use only the supplied background, research question, hypothesis text and your prior scientific knowledge. You have no tools or internet access. Experimental outcomes are unknown to you.
Forecast experimental support for each hypothesis's own causal claim in its stated biological system. Return a complete JSON object in this reply.

User message:

BACKGROUND
[BACKGROUND]

RESEARCH QUESTION
[RESEARCH QUESTION]

CANDIDATE HYPOTHESES
[H1] [FULL HYPOTHESIS 1]

[H2] [FULL HYPOTHESIS 2]

[H3] [FULL HYPOTHESIS 3]

[H4] [FULL HYPOTHESIS 4]

[H5] [FULL HYPOTHESIS 5]

Assess EVERY hypothesis independently. Several different mechanisms may be supported, and none may be supported. There is no required number of recommendations and no requirement to recommend one hypothesis per question.

For each hypothesis estimate p_experimental_support between 0 and 1: your probability that a well-controlled discriminating experiment, competently performed in the species, tissue and scope stated in the hypothesis, would support its central causal claim. Evaluate that claim itself, including a plausible alternative mechanism. This is a subjective scientific forecast, not an observed frequency. If the supplied information cannot justify any numerical estimate, return null and explain the missing information.

Give recommend_for_testing as a separate boolean: would you advise a researcher to spend experimental resources testing this hypothesis now, given its scientific support, testability, assumptions and contradictions? You may recommend multiple hypotheses or none. A recommendation is not a guarantee of success. A numerical estimate is required for a positive recommendation. The comparison will retain your recommendation exactly as issued.

Use the existing scientific considerations: a specific causal chain; consistency with established biology; a feasible discriminating test; fit to the stated question and system; few unsupported assumptions. Novelty is descriptive and is not evidence for or against a claim. Ignore prose style, length, tone, gene-name density and the anonymous candidate order. Do not guess the contents of an undisclosed article or predict another evaluator's score.

Provide a short scientific justification. Quote only literal fragments of the supplied BACKGROUND in support_from_background, or use an empty array. Keep additional assumptions and contradictions separate. Name a concrete intervention, measured readout and necessary controls, and specify outcomes supporting and refuting the claim. These are proposed observations, not results you have seen. Do not announce research, search for information or describe work you will do later; give the complete forecasts now.

Return exactly this structure, with every supplied candidate once:
{"protocol":"experimental-support-v1","forecasts":[{"id":"H1","p_experimental_support":0.5,"recommend_for_testing":false,"support_from_background":[],"assumptions":[],"contradictions":[],"reason":"Short justification","test":{"intervention":"...","measurement":"...","controls":"...","supports_if":"...","refutes_if":"..."}}]}

B.2. The short prompt for five hypotheses

System prompt:

Return a JSON object.

User message:

For each hypothesis below, will it be confirmed experimentally?

BACKGROUND
[BACKGROUND]

RESEARCH QUESTION
[RESEARCH QUESTION]

HYPOTHESES
[H1] [FULL HYPOTHESIS 1]

[H2] [FULL HYPOTHESIS 2]

[H3] [FULL HYPOTHESIS 3]

[H4] [FULL HYPOTHESIS 4]

[H5] [FULL HYPOTHESIS 5]

Return a JSON object with a "forecasts" array. For each hypothesis include its "id" and a boolean "confirmed".

B.3. The short prompt for one statement

System prompt:

Return a JSON object.

User message:

Will this hypothesis be confirmed experimentally?

HYPOTHESIS
[H000000000001] [FULL HYPOTHESIS]

Return a JSON object with a "forecasts" array containing this hypothesis's "id" and a boolean "confirmed".

Appendix C. Strict threshold for matching the reference finding§

The outcome was positive if at least two of the three judges from other developers scored the hypothesis 4 or 5. Each cell gives the percentage, the numerator/denominator, and the 95% confidence interval (CI) from 10,000 resamples of tasks. Match rates under the strict threshold were calculated separately from those under the primary threshold (scores of 3 to 5).

When no hypothesis in the observed set matches the reference finding, the resampling percentile method gives a degenerate interval of 0.0 to 0.0. Such an interval reflects the composition of the observed set and gives no upper bound for the match rate in new tasks.

Model▲Generation method▲Score of 4 or 5 from a majority of judges (95% CI)▲
GPT-6 AstraSingle request1.6% (4/250; 95% CI [0.4, 3.2])
GPT-6 AstraMultistep pipeline1.2% (3/250; 95% CI [0.0, 2.8])
GPT-6 AstraOmega Point1.2% (3/250; 95% CI [0.0, 3.2])
Grok 4.5Single request2.8% (7/250; 95% CI [1.2, 4.8])
Grok 4.5Multistep pipeline4.0% (10/250; 95% CI [1.2, 7.2])
Grok 4.5Omega Point0.0% (0/250; 95% CI [0.0, 0.0])
Claude Opus 5.5Single request2.0% (5/250; 95% CI [0.4, 4.0])
Claude Opus 5.5Multistep pipeline1.2% (3/250; 95% CI [0.0, 3.2])
Claude Opus 5.5Omega Point0.0% (0/250; 95% CI [0.0, 0.0])
Gemini 4 ArgonSingle request3.6% (9/250; 95% CI [1.2, 6.4])
Gemini 4 ArgonMultistep pipeline3.2% (8/250; 95% CI [0.8, 6.0])
Gemini 4 ArgonOmega Point2.0% (5/250; 95% CI [0.0, 4.4])

Appendix D. Full metrics for the prompt comparison§

For each pair of approaches, four forecast metrics (positive predictive value, PPV; negative predictive value, NPV; sensitivity; specificity) were calculated on the same hypotheses; these shared sets are the same as in Table 4. All metrics are given with 95% CIs. A hypothesis that did not match the reference finding counted as a negative outcome.

Predictor and prompt▲PPV▲NPV▲Sensitivity▲Specificity▲
Claude Sonnet 5.5; the detailed prompt20.6% (26/126; 95% CI [13.2, 28.9])91.6% (109/119; 95% CI [86.0, 96.6])72.2% (26/36; 95% CI [56.8, 87.5])52.2% (109/209; 95% CI [46.3, 58.1])
Claude Sonnet 5.5; the short prompt28.6% (14/49; 95% CI [16.7, 40.9])88.8% (174/196; 95% CI [84.0, 93.3])38.9% (14/36; 95% CI [27.0, 52.0])83.3% (174/209; 95% CI [80.5, 86.1])
GPT-6 Astra; the detailed prompt16.0% (32/200; 95% CI [10.1, 22.6])92.0% (46/50; 95% CI [85.2, 98.0])88.9% (32/36; 95% CI [75.8, 97.6])21.5% (46/214; 95% CI [16.5, 26.9])
GPT-6 Astra; the short prompt30.7% (23/75; 95% CI [20.0, 41.8])92.6% (162/175; 95% CI [87.9, 96.6])63.9% (23/36; 95% CI [48.8, 79.3])75.7% (162/214; 95% CI [70.7, 80.5])
Gemini 4 Argon; the detailed prompt21.1% (32/152; 95% CI [13.4, 29.6])95.9% (94/98; 95% CI [91.8, 99.0])88.9% (32/36; 95% CI [77.8, 97.5])43.9% (94/214; 95% CI [38.6, 49.3])
Gemini 4 Argon; the short prompt33.3% (21/63; 95% CI [21.8, 45.6])92.0% (172/187; 95% CI [87.2, 96.2])58.3% (21/36; 95% CI [43.2, 75.0])80.4% (172/214; 95% CI [74.9, 85.0])
Grok 4.5; the detailed prompt19.7% (27/137; 95% CI [12.2, 27.9])95.5% (84/88; 95% CI [90.7, 98.9])87.1% (27/31; 95% CI [73.3, 97.1])43.3% (84/194; 95% CI [39.1, 47.5])
Grok 4.5; the short prompt23.5% (20/85; 95% CI [14.1, 34.0])92.1% (129/140; 95% CI [87.4, 96.3])64.5% (20/31; 95% CI [48.1, 80.0])66.5% (129/194; 95% CI [59.8, 73.0])

In an extended run, further Claude versions also received the detailed prompt. Each predictor’s metrics are calculated over all its usable data, so the denominators may differ from those in the paired comparison above, and the intervals, calculated in a separate resampling, may differ in the last digit. The table lists the predictors that returned usable responses.

Predictor▲Usable hypotheses▲PPV of recommendations (95% CI)▲NPV of negative recommendations (95% CI)▲
Claude Sonnet 5.525020.3% (26/128; 95% CI [12.8, 28.6])91.8% (112/122; 95% CI [86.3, 96.7])
Claude Opus 5.521521.4% (22/103; 95% CI [12.0, 31.8])93.8% (105/112; 95% CI [89.2, 97.5])
Claude Haiku 5.525018.3% (20/109; 95% CI [9.6, 28.1])88.7% (125/141; 95% CI [83.2, 93.7])
Claude Opus 4.824020.9% (28/134; 95% CI [13.4, 29.0])94.3% (100/106; 95% CI [89.1, 99.0])
GPT-6 Astra25016.0% (32/200; 95% CI [10.0, 22.7])92.0% (46/50; 95% CI [85.2, 98.0])
Gemini 4 Argon25021.1% (32/152; 95% CI [13.5, 29.5])95.9% (94/98; 95% CI [91.8, 99.0])
Grok 4.522519.7% (27/137; 95% CI [12.1, 27.9])95.5% (84/88; 95% CI [90.7, 98.9])

Appendix E. Forecasting on the full corpus of 2997 hypotheses§

The outcome was determined for each hypothesis regardless of how it was generated: it was positive if a majority of the judges scored the hypothesis 3 or higher. The self-forecast row gives the forecast of the model that generated the hypothesis. The majority and unanimity rules combined the self-forecast with the forecasts of all external predictors available for that hypothesis, whose number depended on the generator. For the manuscript, the intervals were recalculated by resampling the 50 tasks, keeping all hypotheses of each sampled task together. These descriptive results come from the internal development of the rule.

Forecast rule on 2997 hypotheses▲PPV (95% CI)▲Sensitivity (95% CI)▲
The random control12.1% (179/1477; 95% CI [8.5, 16.1])50.0% (179/358; 95% CI [44.1, 55.4])
Generator forecasts its own hypotheses26.8% (164/611; 95% CI [18.5, 35.6])45.8% (164/358; 95% CI [38.7, 52.4])
Majority of predictors forecast “yes”37.8% (123/325; 95% CI [26.5, 49.2])34.4% (123/358; 95% CI [28.5, 39.5])
All predictors forecast “yes”50.0% (55/110; 95% CI [34.0, 64.2])15.4% (55/358; 95% CI [10.5, 20.3])

Appendix F. Full intervals for four-model unanimity§

The sets and outcome definitions are those of Table 6. Intervals come from 10,000 joint resamples of whole tasks (random seed 20261026). The predictor panel and the random control were analysed with identical formulas and denominators.

Generator and rule▲PPV▲NPV▲Sensitivity▲Specificity▲
Grok 4.5; four-model unanimity73.7% (14/19; 95% CI [52.6, 93.3])88.2% (195/221; 95% CI [83.4, 92.7])35.0% (14/40; 95% CI [25.0, 46.2])97.5% (195/200; 95% CI [95.3, 99.5])
Grok 4.5; the random control17.9% (22/123; 95% CI [10.2, 26.4])84.6% (99/117; 95% CI [78.0, 90.6])55.0% (22/40; 95% CI [40.0, 69.2])49.5% (99/200; 95% CI [43.1, 56.1])
Gemini 4 Argon; four-model unanimity47.4% (9/19; 95% CI [25.0, 70.6])89.6% (207/231; 95% CI [85.9, 93.1])27.3% (9/33; 95% CI [13.6, 42.9])95.4% (207/217; 95% CI [92.8, 97.7])
Gemini 4 Argon; the random control12.1% (16/132; 95% CI [7.0, 17.7])85.6% (101/118; 95% CI [78.7, 91.8])48.5% (16/33; 95% CI [30.6, 66.7])46.5% (101/217; 95% CI [39.4, 53.7])
Claude Opus 5.5; four-model unanimity63.6% (14/22; 95% CI [42.9, 82.4])86.4% (197/228; 95% CI [81.7, 90.8])31.1% (14/45; 95% CI [18.4, 43.9])96.1% (197/205; 95% CI [93.4, 98.5])
Claude Opus 5.5; the random control16.8% (20/119; 95% CI [9.7, 24.6])80.9% (106/131; 95% CI [74.5, 87.2])44.4% (20/45; 95% CI [28.9, 58.8])51.7% (106/205; 95% CI [44.9, 58.5])
Claude Opus 5.5, repeat generation; four-model unanimity58.8% (10/17; 95% CI [35.0, 81.8])85.5% (195/228; 95% CI [80.2, 90.3])23.3% (10/43; 95% CI [12.5, 34.7])96.5% (195/202; 95% CI [94.0, 98.6])
Claude Opus 5.5, repeat generation; the random control18.8% (25/133; 95% CI [11.7, 26.8])83.9% (94/112; 95% CI [77.5, 90.0])58.1% (25/43; 95% CI [44.1, 71.1])46.5% (94/202; 95% CI [40.4, 52.6])

Appendix G. Equal-length lists drawn from single-model forecasts§

This calculation asks how many matching hypotheses would remain if a single model’s recommendations were cut at random to the length of the predictor panel’s list. For example, on the Grok hypotheses GPT-6 Astra made 53 positive forecasts, 25 of which match the reference finding. A random 19 of these 53 contain on average 8.96 matching hypotheses (47.2% of the list), with a central 95% range of 6 to 12. The predictor panel selected 19 hypotheses, 14 of them matching (73.7%; Appendix M.6).

For each generator and model, the table gives the pool of positive forecasts, the length of the random list, and its expected result. All hypotheses in a pool are equally likely to be drawn, and none is drawn twice. The “Full set” row repeats the baseline random control of Table M.6. Ranges come from the exact distribution, with hypotheses and judge scores held fixed. These additional calculations describe how the previously chosen rule performs on the same data.

Generator▲Pool from which hypotheses are drawn▲Matching the reference finding / total in pool▲Hypotheses in random list▲Random selection, mean number matching the reference finding▲Random selection, central 95% range of the number matching the reference finding▲
Grok 4.5Full set40/240193.17 out of 19 (16.7%)0 to 6
Grok 4.5“Yes” forecasts from Claude Sonnet 5.521/46198.67 out of 19 (45.7%)5 to 12
Grok 4.5“Yes” forecasts from GPT-6 Astra25/53198.96 out of 19 (47.2%)6 to 12
Grok 4.5“Yes” forecasts from Gemini 4 Argon23/52198.40 out of 19 (44.2%)5 to 12
Grok 4.5“Yes” forecasts from Grok 4.531/110195.35 out of 19 (28.2%)2 to 9
Gemini 4 ArgonFull set33/250192.51 out of 19 (13.2%)0 to 6
Gemini 4 Argon“Yes” forecasts from Claude Sonnet 5.512/45195.07 out of 19 (26.7%)2 to 8
Gemini 4 Argon“Yes” forecasts from GPT-6 Astra14/51195.22 out of 19 (27.5%)2 to 8
Gemini 4 Argon“Yes” forecasts from Gemini 4 Argon18/53196.45 out of 19 (34.0%)3 to 10
Gemini 4 Argon“Yes” forecasts from Grok 4.520/105193.62 out of 19 (19.0%)1 to 7
Claude Opus 5.5Full set45/250223.96 out of 22 (18.0%)1 to 8
Claude Opus 5.5“Yes” forecasts from Claude Sonnet 5.519/50228.36 out of 22 (38.0%)5 to 12
Claude Opus 5.5“Yes” forecasts from GPT-6 Astra21/49229.43 out of 22 (42.9%)6 to 13
Claude Opus 5.5“Yes” forecasts from Gemini 4 Argon22/56228.64 out of 22 (39.3%)5 to 12
Claude Opus 5.5“Yes” forecasts from Grok 4.526/110225.20 out of 22 (23.6%)2 to 9

Appendix H. Proportion of correct forecasts in the 50/50 set§

The original protocol specified balanced accuracy, the mean of sensitivity and specificity, as the primary metric. Because the set contains equal numbers of confirmed and refuted statements, balanced accuracy here equals the proportion of correct forecasts. The nominal 95% CIs are taken from the report and come from 10,000 resamples of articles, each resampled with both its statements. Under unanimity, a statement receives a final “yes” only if all five forecasts are positive; every other statement receives a final “no”. A high proportion of confirmed statements among those selected unanimously therefore comes with many missed confirmed statements and a lower overall proportion of correct forecasts.

Model or rule▲Proportion of correct forecasts on the 50/50 set (95% CI)▲
Claude Sonnet 5.574.0% (95% CI [66.0, 82.0])
GPT-6 Astra71.0% (95% CI [63.0, 79.0])
Gemini 4 Argon78.0% (95% CI [69.0, 86.0])
Grok 4.575.0% (95% CI [65.0, 84.0])
Claude Opus 5.573.0% (95% CI [66.0, 80.0])
Five-model unanimity70.0% (95% CI [63.0, 77.0])
The random control52.0% (95% CI [43.0, 61.0])

Appendix I. Search query and publication selection log§

The query is preserved in the original export of 13 October 2026. The edat field gives the date a record entered PubMed; each article’s eligibility was also checked against the date of its first public version and against any preprints.

(aging[MeSH Terms] OR ageing[tiab] OR aging[tiab] OR longevity[tiab] OR lifespan[tiab] OR senescence[tiab] OR healthspan[tiab]) AND ("Nature"[ta] OR "Science"[ta] OR "Cell"[ta] OR "Nat Aging"[ta] OR "Cell Metab"[ta] OR "Nat Med"[ta] OR "Nat Cell Biol"[ta] OR "Cell Stem Cell"[ta] OR "Aging Cell"[ta] OR "Nat Commun"[ta] OR "Sci Adv"[ta] OR "Proc Natl Acad Sci U S A"[ta] OR "Mol Cell"[ta] OR "Immunity"[ta] OR "Neuron"[ta]) AND journal article[pt] NOT review[pt] NOT clinical trial[pt] AND ("2026/08/01"[edat] : "2026/10/12"[edat])

The recorded sequence comprises 284 retrieved records, 72 exclusions at the preliminary filter, and 212 provisionally eligible candidates; the final set has 50 tasks. The extraction log keeps only the last run for each article, because earlier runs were overwritten when articles were replaced; its count of records with the status “accepted” is therefore not a stage of the selection in Figure 2. The selection log gives the identifiers of the final tasks, the order of the 212 candidates, the reasons for the 72 preliminary exclusions, and the checksums of the source files. Selecting tasks for suitability and for a concealed reference finding limits how representative the set is.

Appendix J. Agreement among judges in binary votes on matching the reference finding§

A judge’s vote was positive if the judge’s score reached the threshold: 3 for the primary threshold and 4 for the strict threshold. The first table compares the four judges pairwise across all 2997 generated hypotheses. These comparisons include the scores of the judge from the generator’s developer, which were excluded when the outcome was determined. “First” and “second” judge in the column headers follow the order of the names in the first column. Hypothesis counts and the proportion of agreeing votes refer to the vote “score of 3 or higher”. Cohen’s kappa is 1 for complete agreement and 0 for the agreement expected from independent votes with each judge’s observed proportion of positive votes. Quadratically weighted kappa compares scores across the full 0 to 5 scale. The 95% confidence intervals come from 10,000 resamples of the 50 tasks.

Judge pair▲Hypotheses with a score of 3 or higher from both judges▲Hypotheses with a score of 3 or higher from the first judge only▲Hypotheses with a score of 3 or higher from the second judge only▲Hypotheses with a score below 3 from both judges▲Proportion of hypotheses with agreeing votes from the two judges, %▲Cohen’s kappa for the vote “score of 3 or higher” (95% CI)▲Cohen’s kappa for the vote “score of 4 or 5” (95% CI)▲Weighted kappa across the full 0 to 5 scale▲
GPT-5.6 Sol and Grok 4.732515561245692.80.71 (95% CI [0.66, 0.76])0.67 (95% CI [0.50, 0.81])0.68
GPT-5.6 Sol and Claude Opus 5.531416649246892.80.70 (95% CI [0.65, 0.76])0.54 (95% CI [0.39, 0.65])0.45
GPT-5.6 Sol and Gemini 4 Argon31017040247793.00.71 (95% CI [0.64, 0.77])0.61 (95% CI [0.45, 0.73])0.62
Grok 4.7 and Claude Opus 5.52998764254795.00.77 (95% CI [0.73, 0.81])0.47 (95% CI [0.28, 0.60])0.38
Grok 4.7 and Gemini 4 Argon28110569254294.20.73 (95% CI [0.67, 0.78])0.72 (95% CI [0.58, 0.80])0.52
Claude Opus 5.5 and Gemini 4 Argon2758875255994.60.74 (95% CI [0.69, 0.78])0.60 (95% CI [0.44, 0.71])0.64

The second table includes, for each hypothesis, only the three judges from other developers, whose votes determined its outcome. The outcome is positive if two or three judges gave a score in the range of the first column. Fleiss’ kappa generalises Cohen’s kappa to three judges.

Judge score▲Hypotheses with this score from all three judges▲Hypotheses with this score from two of the three judges▲Hypotheses with this score from one of the three judges▲Hypotheses with this score from none of the judges▲Fleiss’ kappa for three judges (95% CI)▲
3 or higher2639518824510.72 (95% CI [0.67, 0.77])
4 or 536217328670.57 (95% CI [0.41, 0.67])

Appendix K. Additional calculations for selection and forecasts§

These calculations use the recorded run reports and make no new model calls. Intervals come from 10,000 resamples of whole tasks (random seed 20261030). The methods are described in Section 2.10 and Appendix L.6.

K.1. Ranking hypotheses by the numerical probability of confirmation

This calculation uses the 250 hypotheses that GPT-6 Astra generated in a single request and the probability each predictor assigned to them under the detailed prompt. The area under the ROC curve (AUC) is the probability that a hypothesis matching the reference finding receives a higher score than a non-matching one, with ties counted as 0.5. Top-k precision is the expected number of matches divided by the list length; when probabilities are tied at the list boundary, the expectation uses the match rate within the tied group. The baseline match rate is 14.4% (36/250).

Predictor▲AUC (95% CI)▲Top-19 precision▲Top-20 precision▲Top-21 precision▲Top-22 precision▲
Claude Sonnet 5.50.71 (95% CI [0.62, 0.79])38.9% (7.4/19)38.0% (7.6/20)37.1% (7.8/21)36.4% (8.0/22)
Claude Opus 5.50.71 (95% CI [0.60, 0.79])36.8% (7.0/19)36.7% (7.3/20)36.5% (7.7/21)36.4% (8.0/22)
Claude Haiku 5.50.62 (95% CI [0.50, 0.72])27.6% (5.2/19)27.5% (5.5/20)27.4% (5.8/21)27.3% (6.0/22)
Claude Opus 4.80.67 (95% CI [0.56, 0.77])33.3% (6.3/19)32.5% (6.5/20)31.7% (6.7/21)31.1% (6.8/22)
GPT-6 Astra0.68 (95% CI [0.57, 0.79])42.1% (8.0/19)40.0% (8.0/20)39.3% (8.2/21)38.6% (8.5/22)
Gemini 4 Argon0.74 (95% CI [0.64, 0.82])35.7% (6.8/19)35.0% (7.0/20)35.7% (7.5/21)36.4% (8.0/22)
Grok 4.50.71 (95% CI [0.60, 0.81])36.8% (7.0/19)35.0% (7.0/20)33.3% (7.0/21)31.8% (7.0/22)

Unanimous selection by the four predictors was applied to the same hypotheses, using the four predictors’ recorded answers to the short prompt. All four predictors answered for 245 hypotheses in 49 tasks. The rule selected 22 of them, and 50.0% of the selected hypotheses matched the reference finding (11/22; 95% CI [30.0, 70.4]) against a baseline match rate of 14.7% (36/245), a ratio of 3.40 (95% CI [2.26, 4.95]). The table below compares this list with the 22 hypotheses ranked highest by probability among the same 245. Hypotheses without a numerical probability were excluded from the ranking. The difference is the match rate among unanimously selected hypotheses minus the match rate among the top-ranked hypotheses; the p-value comes from permuting the two lists within tasks, with Holm correction across the seven predictors.

Predictor whose probability ranked the hypotheses▲Hypotheses with a numerical probability▲Match rate among the top 22▲Ratio to the baseline match rate (95% CI)▲Unanimity minus ranking, percentage points (95% CI)▲Holm-adjusted p▲
Claude Sonnet 5.524536.4% (8.0/22)2.47 (95% CI [1.29, 4.00])13.6 (95% CI [−9.1, 36.7])0.6573
Claude Opus 5.521036.4% (8.0/22)2.47 (95% CI [1.25, 3.98])13.6 (95% CI [−7.5, 34.2])0.6573
Claude Haiku 5.524527.3% (6.0/22)1.86 (95% CI [0.70, 3.20])22.7 (95% CI [2.2, 45.2])0.3031
Claude Opus 4.823531.1% (6.8/22)2.11 (95% CI [0.82, 3.54])18.9 (95% CI [−2.9, 42.5])0.6573
GPT-6 Astra24038.6% (8.5/22)2.63 (95% CI [1.01, 4.28])11.4 (95% CI [−11.1, 36.1])0.6573
Gemini 4 Argon24536.4% (8.0/22)2.47 (95% CI [1.15, 4.08])13.6 (95% CI [−2.6, 30.4])0.6573
Grok 4.522031.8% (7.0/22)2.17 (95% CI [0.91, 3.88])18.2 (95% CI [−4.0, 40.0])0.6573

K.2. Predictor panel versus the best single model at equal list length

The best single model is the predictor with the highest match rate among its positive forecasts. Shortening its list at random to the panel’s list length leaves the expected match rate unchanged, so the rates are compared directly. The p-value comes from permuting the two rules within each task, with Holm correction across the four sets. The rule was chosen on the Grok hypotheses, and both rules use the same forecasts.

Hypothesis set▲Match rate among hypotheses selected by the panel▲Best single model▲Match rate among its positive forecasts▲Difference, percentage points (95% CI)▲Holm-adjusted p▲
Grok 4.573.7% (14/19)GPT-6 Astra47.2% (25/53)26.5 (95% CI [8.2, 45.9])p < 0.05
Gemini 4 Argon47.4% (9/19)Gemini 4 Argon34.0% (18/53)13.4 (95% CI [−4.3, 31.5])0.0833
Claude Opus 5.563.6% (14/22)GPT-6 Astra42.9% (21/49)20.8 (95% CI [6.8, 35.6])p < 0.05
Claude Opus 5.5, repeat generation58.8% (10/17)Gemini 4 Argon34.4% (21/61)24.4 (95% CI [6.0, 44.7])p < 0.01

K.3. Selection without the predictor from the generator’s developer

The predictor from the generator’s developer was removed from the panel, and the three remaining predictors still had to agree unanimously. The last column repeats the full-panel result of Table 6.

Hypothesis set▲Excluded predictor▲Match rate among selected hypotheses (95% CI)▲Proportion of all matching hypotheses found▲Match rate with the full panel▲
Grok 4.5Grok 4.568.2% (15/22; 95% CI [47.6, 86.7])37.5% (15/40)73.7% (14/19)
Gemini 4 ArgonGemini 4 Argon45.0% (9/20; 95% CI [23.1, 66.7])27.3% (9/33)47.4% (9/19)
Claude Opus 5.5Claude Sonnet 5.553.6% (15/28; 95% CI [35.0, 70.8])33.3% (15/45)63.6% (14/22)
Claude Opus 5.5, repeat generationClaude Sonnet 5.546.4% (13/28; 95% CI [26.1, 65.6])30.2% (13/43)58.8% (10/17)

K.4. Decisions within pairs in the 50/50 set

Each article in the set contributed one confirmed and one refuted statement. The columns give the four possible decisions on a pair, and together they sum to the number of pairs.

Model or rule▲Article pairs▲Confirmed accepted, reversed rejected▲Both statements accepted▲Both statements rejected▲Reversed accepted, confirmed rejected▲
Claude Sonnet 5.55026 (52.0%)1392
GPT-6 Astra5023 (46.0%)1872
Gemini 4 Argon5031 (62.0%)6103
Grok 4.55030 (60.0%)695
Claude Opus 5.55024 (48.0%)1871
Random control5012 (24.0%)151310

The “effect in the favourable direction” rule labelled 61 of the 100 statements. The proportion of correct answers is reported separately for labelled and unlabelled statements.

Model or rule▲Proportion of correct answers among the 61 labelled statements▲Proportion of correct answers among the 39 unlabelled statements▲
Claude Sonnet 5.568.9% (42/61)82.1% (32/39)
GPT-6 Astra77.0% (47/61)61.5% (24/39)
Gemini 4 Argon75.4% (46/61)82.1% (32/39)
Grok 4.575.4% (46/61)74.4% (29/39)
Claude Opus 5.570.5% (43/61)76.9% (30/39)
Random control52.5% (32/61)51.3% (20/39)
“Effect in the favourable direction” rule65.6% (40/61)No label assigned

The rule labelled both statements in 20 pairs. The table below shows the decisions of the rule, the models, and the random control on these pairs; the rule accepts a statement labelled “favourable” and rejects one labelled “unfavourable”.

Model or rule▲Article pairs▲Confirmed accepted, reversed rejected▲Both statements accepted▲Both statements rejected▲Reversed accepted, confirmed rejected▲
“Effect in the favourable direction” rule2051302
Claude Sonnet 5.5209542
GPT-6 Astra2011540
Gemini 4 Argon2011351
Grok 4.52013241
Claude Opus 5.5208741
Random control205663

K.5. Enrichment under the strict threshold

The strict threshold requires a score of 4 or 5 from a majority of the judges. The ratio is the match rate among selected hypotheses divided by the baseline match rate at the same threshold; the selected lists are unchanged.

Hypothesis set▲Match rate among selected hypotheses▲Baseline match rate▲Ratio of these rates (95% CI)▲Proportion of all matching hypotheses found▲
Grok 4.526.3% (5/19)2.9% (7/240)9.02 (95% CI [4.21, 16.00])71.4% (5/7)
Gemini 4 Argon31.6% (6/19)3.6% (9/250)8.77 (95% CI [5.18, 14.71])66.7% (6/9)
Claude Opus 5.59.1% (2/22)2.0% (5/250)4.55 (95% CI [0.00, 11.03])40.0% (2/5)
Claude Opus 5.5, repeat generation11.8% (2/17)1.2% (3/245)9.61 (95% CI [0.00, 18.85])66.7% (2/3)

K.6. Originality, feasibility, and clarity of selected and excluded hypotheses

Each selected hypothesis was paired with an excluded hypothesis from the same task. A hypothesis’s score is the mean of the three judges from other developers on the 1 to 10 scale. Differences are selected minus excluded; intervals come from 10,000 task resamples.

Hypothesis set▲Metric▲Hypothesis pairs▲Selected, mean score▲Excluded, mean score▲Difference, selected minus excluded (95% CI)▲Pairs with a higher score for the selected hypothesis▲Pairs with a lower score for the selected hypothesis▲Pairs with equal scores▲
Grok 4.5Originality194.494.72−0.23 (95% CI [−0.75, 0.32])7111
Grok 4.5Feasibility196.816.460.35 (95% CI [−0.35, 1.05])865
Grok 4.5Clarity198.147.950.19 (95% CI [−0.04, 0.46])856
Gemini 4 ArgonOriginality194.825.00−0.18 (95% CI [−0.58, 0.26])6103
Gemini 4 ArgonFeasibility196.706.020.68 (95% CI [0.39, 0.98])1315
Gemini 4 ArgonClarity198.047.260.77 (95% CI [0.51, 1.05])1603
Claude Opus 5.5Originality215.245.48−0.24 (95% CI [−0.62, 0.13])8103
Claude Opus 5.5Feasibility216.596.250.33 (95% CI [−0.02, 0.72])1164
Claude Opus 5.5Clarity218.258.060.19 (95% CI [0.05, 0.33])8211
All three sets combinedOriginality594.865.08−0.21 (95% CI [−0.47, 0.04])21317
All three sets combinedFeasibility596.696.240.45 (95% CI [0.17, 0.73])321314
All three sets combinedClarity598.157.770.38 (95% CI [0.24, 0.53])32720

Logistic regression on the 119 hypotheses in this comparison (60 selected and 59 excluded) estimated the association between selection and matching the reference finding after adjustment for clarity. The outcome is 1 if the hypothesis matches the reference finding at the primary threshold. Clarity and feasibility are the mean scores of the three judges, centred on their means across the 119 hypotheses. In total, 37 of the 60 selected and 15 of the 59 excluded hypotheses matched the reference finding. Wald intervals and p-values use standard errors clustered on the 58 generator-by-task combinations; a separate column gives the percentile interval from 10,000 resamples of these combinations.

Variables in the regression▲Variable▲Odds ratio (95% CI)▲Task-resample interval▲p▲
Selection onlySelection (selected versus excluded)4.72 (95% CI [2.36, 9.42])[2.51, 10.30]p < 0.001
Selection and claritySelection (selected versus excluded)3.85 (95% CI [1.81, 8.20])[1.90, 9.04]p < 0.001
Selection and clarityClarity, per 1-point increase2.02 (95% CI [1.04, 3.95])[1.04, 4.42]p < 0.05
Selection, clarity, and feasibilitySelection (selected versus excluded)4.03 (95% CI [1.89, 8.60])[1.96, 9.90]p < 0.001
Selection, clarity, and feasibilityClarity, per 1-point increase2.14 (95% CI [1.05, 4.34])[1.06, 4.99]p < 0.05
Selection, clarity, and feasibilityFeasibility, per 1-point increase0.88 (95% CI [0.57, 1.36])[0.56, 1.48]0.5647
Clarity onlyClarity, per 1-point increase2.75 (95% CI [1.56, 4.88])[1.62, 5.41]p < 0.001

K.7. Repeat run of the forecasts

The comparison covers hypotheses that received forecasts in both runs. Cohen’s kappa (κ) was calculated on each model’s “yes” and “no” decisions. The match rate among hypotheses selected in the initial run is reported for the same hypotheses, so its denominator can differ from Table 6.

Hypothesis set▲Hypotheses with forecasts in both runs▲Decision agreement, Claude Sonnet 5.5▲Decision agreement, GPT-6 Astra▲Decision agreement, Gemini 4 Argon▲Decision agreement, Grok 4.5▲Selected in the initial run▲Selected in the repeat run▲Selected in both runs▲Match rate among hypotheses selected in the repeat run (95% CI)▲Match rate among hypotheses selected in the initial run▲
Grok 4.523593.2% (219/235; κ = 0.78)94.0% (221/235; κ = 0.83)96.2% (226/235; κ = 0.89)91.9% (216/235; κ = 0.84)18191668.4% (13/19; 95% CI [46.7, 88.9])72.2% (13/18)
Gemini 4 Argon24593.5% (229/245; κ = 0.78)95.1% (233/245; κ = 0.85)95.5% (234/245; κ = 0.86)88.6% (217/245; κ = 0.76)19181750.0% (9/18; 95% CI [26.7, 75.0])47.4% (9/19)
Claude Opus 5.524090.8% (218/240; κ = 0.71)96.2% (231/240; κ = 0.88)93.8% (225/240; κ = 0.83)88.3% (212/240; κ = 0.76)21211566.7% (14/21; 95% CI [43.5, 86.4])61.9% (13/21)

K.8. Lexical check of key elements

An element of the reference finding counted as named if at least two-thirds of its content words appeared in the hypothesis text. The calculation covers the 1000 single-request hypotheses. The AUC measures how well the share of named elements separates hypotheses that the judges scored as matching the reference finding from those they did not; its interval comes from 10,000 task resamples. Among the 60 hypotheses selected unanimously by the four predictors, matching hypotheses named on average 17.3% of the elements and the remaining hypotheses 6.1%; at least two elements were named in 6 of the 37 matching hypotheses and in 2 of the 23 others. Among all 1000 hypotheses, at least one element was named in 46.2% (72/156) of matching and 16.4% (138/844) of non-matching hypotheses (odds ratio 4.39, 95% CI [2.87, 6.81]).

Generator▲Hypotheses▲Matching the reference finding▲AUC of the share of named elements▲
Claude Opus 5.5250450.62
Gemini 4 Argon250330.76
GPT-6 Astra250360.63
Grok 4.5250420.63
All four models combined10001560.65 (95% CI [0.60, 0.70])

Appendix L. Additional methodological details§

L.1. Model run settings

The token limit applies to one model call and covers both reasoning and the response. Response attempts give how many times a request was repeated when the response could not be parsed or the model refused to answer.

Role▲Models▲System prompt▲Token limit per call▲Response attempts▲Tools and internet access▲Reasoning setting▲
Hypothesis generatorsGPT-6 Astra, Grok 4.5, Claude Opus 5.5, Gemini 4 Argonthe task, without the reference finding or key elements32,000up to three per single request and up to three per pipeline stepdisabled; not confirmed per request for the original generations (Section 2.9)high reasoning effort
JudgesGPT-5.6 Sol, Grok 4.7, Claude Opus 5.5, Gemini 4 Argonthe reference finding, the key elements, and the matching scale32,000up to threedisabled; not confirmed per requestreasoning = medium
Predictors, detailed promptClaude Sonnet 5.5, GPT-6 Astra, Gemini 4 Argon, Grok 4.5; in the extended run, also Claude Opus 5.5, Claude Haiku 5.5, and Claude Opus 4.8scientific criteria and an instruction to return JSON (Appendix B.1)32,000twodisabled; confirmed from logs for the runs of 22 to 24 Octoberhigh reasoning effort
Predictors, short promptClaude Sonnet 5.5, GPT-6 Astra, Gemini 4 Argon, Grok 4.5, Claude Opus 5.5an instruction to return JSON (Appendices B.2 and B.3)32,000twodisabled; confirmed from logshigh reasoning effort

L.2. Vote distribution and judge agreement across the full scale

All three judges scored 263 hypotheses 3 or higher; two of the three judges did so for 95 hypotheses, one judge for 188, and no judge for 2451. A single changed vote would flip the majority decision for 283 hypotheses: 95 were classified as matching by two votes of three, and 188 as non-matching with one vote in favour. Across individual judge pairs, the bounds of the 95% CI for Cohen’s kappa on the “score of 3 or higher” vote ranged from 0.64 to 0.81.

Quadratically weighted kappa counts disagreements across the whole 0 to 5 scale, including those within the 0 to 2 and 3 to 5 ranges, which do not change the outcome at the primary threshold. For every judge pair it was lower than Cohen’s kappa for the “score of 3 or higher” vote, ranging from 0.38 to 0.68; Spearman’s rank correlation between scores ranged from 0.57 to 0.66. Appendix J gives the weighted kappa for each judge pair.

L.3. The random control and equal-length random lists

Random answers were generated with the Node.js crypto.randomInt function for the 2997-hypothesis set, the unanimous-selection sets, and the Claude Opus 5.5 repeat generation, and with crypto.randomBytes for the 250 GPT-6 Astra hypotheses and the 50/50 set. Forecasting on the full corpus of 2997 hypotheses used a separate recorded sequence with 1477 “yes” answers.

To control for list length, we considered drawing, with equal probability and without replacement, exactly as many distinct hypotheses as the predictor panel selected: 19 of the 240 Grok hypotheses (40 of which match the reference finding), 19 of the 250 Gemini hypotheses, and 22 of the 250 Opus hypotheses. The program computed the exact hypergeometric distribution of the number of matches in such a list, its mean, and its central 95% range; the mean was also checked against 100,000 random draws from this distribution. The calculation needs only the set size, the number of matching hypotheses in the set, and the list length; the simulations did not sample hypothesis identifiers.

For comparison with a single model, that model’s positive forecasts were randomly subsampled to the panel’s list length. For example, GPT-6 Astra answered “yes” for 53 Grok hypotheses, 25 of which match the reference finding; we calculated the result of drawing 19 of those 53 at random. This control was run for each of the four predictors and each generator. All hypotheses in the “yes” group had equal probability, because binary answers define only a “yes” group and a “no” group. Appendix G gives the results.

L.4. Enumeration of selection rules

The enumeration of 80 voting rules on the Grok hypotheses tested, for each combination of k models, all k thresholds on the number of “yes” answers: 5 rules for single models, 20 for pairs, 30 for triples, 20 for quadruples, and 5 for the full panel. The final four-model unanimity rule is one of these 80 rules.

In the 32 two-stage schemes, one “yes” answer was enough to pass the second stage. In 22 of them, the first-stage model was also on the second-stage panel, so its own “yes” already passed the second stage; these schemes duplicated selection by a single model, and the remaining 10 defined additional rules.

The sets and the rule for the Gemini 4 Argon and Claude Opus 5.5 hypotheses were fixed on 23 October 2026 at 21:43 and 22:09 UTC, respectively, before the first requests to the predictors.

L.5. Isolation of the initial Claude Opus 5.5 generation

The benchmark script requested that tools be disabled, but until a fix on 21 October the tool-disabling parameter was lost when requests were streamed. Request logs for 13 to 18 October were not preserved. On 27 October this configuration was reproduced with the same software version and settings, and the model was asked to open a web page, run a web search, and read the task file containing the reference findings. All five of the model’s tool calls were rejected; no web search or page load took place. A search of the 685 saved raw responses of the initial generation and the 647 of the repeat generation found no URLs, DOIs, PubMed identifiers, mentions of the year 2026, preprint servers, or the DOI of the task article. The same search of the 2916 saved raw responses of the four generators also found none.

In the repeat generation of 27 to 28 October, each of the 792 successful requests was matched to the call log and to a saved record confirming that tools were disabled. The same three judges from other developers scored the hypotheses with the same prompt. If the Claude safety classifier cut off the response on every attempt and no hypotheses remained, the set was counted as a refusal, that is, as five non-matching hypotheses. The multistep pipeline set for task R42736449 was not run on 27 October: its number of generated tokens is matched to Omega Point on the same task, and the script did not recognise the Claude refusal in Omega Point. The set was generated on 28 October after the script was fixed.

L.6. Statistical procedures

Random seeds. Generation intervals used random seed 20261025; intervals for unanimous selection and for the random control on the same hypotheses (Table 6, Appendix F) used seed 20261026. These intervals replace those in the initial reports, which were based on 2000 resamples. The sensitivity analyses and the cueing analysis used seed 20261027, and the AUC intervals used 20261030. Generators were compared across the 48 shared tasks with 10,000 joint resamples.

Family of 150 originality comparisons. Five metrics were evaluated: originality, technical feasibility, clarity, distinctiveness of the two ideas within one response, and an overall score, the mean of those four ratings. For each metric, we compared three pairs of generation methods within each of the four models (4 × 3 × 5 = 60) and six pairs of models within each of the three methods (6 × 3 × 5 = 90). Each pairwise comparison used the judges common to both models, excluding the judges from both models’ developers. Adjustments for individual judges and for all judges combined were calculated separately and are not part of the family of 150 comparisons.

Sensitivity analyses. The overall match rate in single-request generation and the match rate among selected hypotheses in the final selection were recalculated on the tasks with a cueing score of 0 or 1. The final selection was also re-evaluated leaving out one judge at a time; a positive outcome then required a score of 3 or higher from both remaining judges, and the selected lists were unchanged. Opus generation without the sets containing a Claude refusal was recalculated with the parameters of the primary analysis and Holm correction across the three within-model comparisons.

Probability ranking. For the 250 hypotheses that Astra generated in a single request, we calculated, for each predictor under the detailed prompt, the AUC of the confirmation probability and the match rate among the top k ranked hypotheses for k = 19 to 22, the lengths of the final unanimous-selection lists. When probabilities were tied at the list boundary, the expected number of matches was calculated from the match rate within the tied group. Unanimous selection by the four predictors was applied to their saved answers to the short prompt for 245 hypotheses in 49 tasks, and its list was compared with each model’s top k ranked hypotheses, k being the length of the rule’s list. The difference in match rates was tested by permuting the two lists within tasks, with Holm correction across the seven models.

Comparison with the best single model. The best model is the one with the highest match rate among its “yes” forecasts. Randomly subsampling its list to the panel’s list length leaves the expected match rate among selected hypotheses unchanged, so the rates themselves were compared. Two-sided p-values come from permuting the two rules within each task, by exhaustive enumeration when few tasks differed and otherwise by 19,999 random permutations with one added to both numerator and denominator; Holm correction was applied across the four sets. The panel’s list is a subset of its members’ “yes” forecasts, so the two rules are dependent.

Selection without the predictor from the generator’s developer. The predictor from the generator’s developer was removed from the panel, and the three remaining predictors had to agree unanimously.

Within-pair decisions on the 50/50 set. For each article, we checked whether the model accepted the confirmed statement and rejected the refuted one. The “effect in the favourable direction” control is a deterministic rule applied to the statement text: the direction of change follows from the verb and from the sign that the measured indicator has for the organism. Statements about indicators with no agreed sign were left unlabelled, and the labelling did not use the experimental labels. Model accuracy was calculated separately for labelled and unlabelled statements.

Enrichment at the strict threshold. We divided the match rate among selected hypotheses by the baseline match rate at the same threshold; the interval comes from the same task resamples.

Lexical check of key elements. In the 1000 single-request hypotheses, an element of the reference finding counted as named if at least two-thirds of its content words appeared in the hypothesis text. The check detects shared words and does not count paraphrase. The association between the share of named elements and the judges’ decision was measured by the AUC and by the odds ratio for naming at least one element; intervals come from 10,000 task resamples. This check does not replace the judges’ evaluation.

Repeat run of the forecasts. On 30 October, the four predictors received requests byte-identical to those of 23 October: the cohorts, the random-control answers, and all 600 request items matched. Each request’s isolation was verified against the call log and the records confirming that tools were disabled; one model ran on three sets concurrently, and the log was cross-checked with those concurrent runs taken into account. Repeatability was measured by the proportion of agreeing decisions, Cohen’s kappa, and the number of hypotheses selected in both runs.

Originality of selected and excluded hypotheses. Each selected hypothesis was paired with one excluded hypothesis from the same task, chosen by hash. Three judges from other developers scored both with the prompt of the originality experiment: originality, feasibility, and clarity on a 1 to 10 scale, one hypothesis per request, with no information about selection. The three judges’ scores were averaged; the “selected minus excluded” difference was evaluated with 10,000 task resamples and within-task sign permutation (19,999 permutations), with Holm correction across the three metrics. The association between selection and matching after adjustment for clarity was estimated by logistic regression of a match with the reference finding at the primary threshold on 119 hypotheses; the independent variables were membership in the selected set and the mean clarity score, and standard errors were clustered on the generator-by-task combinations.

Degree of cueing. For each task, the match rates of the four generators in single-request generation were averaged; the task was the unit of analysis (50 tasks, 1000 hypotheses). Two-sided p-values were based on the absolute correlation over 99,999 permutations of question scores between whole tasks, with one added to both numerator and denominator. Confidence intervals come from 10,000 resamples of whole tasks, with ranks recalculated in each. Exploratory checks included a joint rating of the research question and the background, other generation methods, alternative ratings of borderline questions, and exclusion of previously discussed examples. The annotations, data, and parameters are given in the report on cueing in the tasks; this analysis did not use model forecasts.

L.7. Order of candidate review

Candidates were ordered by the SHA-256 hash of the string omega-creativity-2026-10-11 followed by the PubMed identifier. This ordering reproduces the saved order of the 212 candidates.

L.8. Definitions of measures

  • overall match rate: the proportion of all hypotheses in a set that match the reference finding;
  • positive predictive value (PPV): the proportion of matching hypotheses among those selected or forecast “yes” (in the 50/50 set, the proportion of confirmed statements);
  • negative predictive value (NPV): the proportion of non-matching hypotheses among those forecast “no” (in the 50/50 set, the proportion of refuted statements);
  • sensitivity: the proportion of matching hypotheses (confirmed statements) that were selected or forecast “yes”;
  • specificity: the proportion of non-matching hypotheses (refuted statements) forecast “no”;
  • balanced accuracy: the mean of sensitivity and specificity;
  • area under the ROC curve (AUC): the probability that a randomly chosen matching hypothesis receives a higher score than a randomly chosen non-matching one.

Appendix M. Additional results§

M.1. Claude Opus 5.5 repeat generation and Claude refusals

With single-request generation, the match rate in the repeat generation was higher than in the initial generation in 11 tasks, lower in 13, and the same in 26. With the Omega Point method, the match outcome differed in eight of the fifty tasks; exhaustive enumeration of within-task permutations gives p = 0.0625 before correction and 0.188 after, while the smallest value attainable with this task composition is 0.0234. The confidence interval estimates the difference under task resampling, whereas the permutation test checks whether the two generations are exchangeable within each task.

The repeat-generation match rates include two sets that Opus refused to generate: the multistep pipeline for task R42664348 and the Omega Point method for task R42736449. In both, the classifier cut off the response at the hypothesis-generation stage in all six attempts over two runs (three per run), after which the model refused to try again. Without these sets, the overall match rate was 14.7% (36/245) with the multistep pipeline and 1.2% (3/245) with the Omega Point method. Retained sets also contained refusals at individual stages: 10 multistep-pipeline sets and 10 Omega Point sets in the initial generation, and 7 and 2 sets in the repeat generation; in these, the next stage received a model refusal or a truncated response instead of a result. In two single-request responses in the repeat generation, the model rewrote the complete set of hypotheses after a cutoff. In the repeat generation, the multistep pipeline differed from single-request generation by −3.6 percentage points (95% CI [−8.4, 1.2]; p = 0.194 after Holm correction across three within-model comparisons).

A separate analysis excluded every set in which the saved response of at least one stage contained a Claude refusal or a fragment truncated by the classifier: 10 multistep-pipeline sets and 10 Omega Point sets in the initial generation, and 8 multistep-pipeline sets, 3 Omega Point sets, and 2 single-request sets in the repeat generation, including both sets that failed on every attempt. Before the fix of 14 October, the truncated fragment was saved together with the retry response; a cutoff followed by a complete response leaves no trace in the saved text. The repeat-generation log records 103 message cutoffs by the classifier, including those in the excluded sets. Without the excluded sets, the match rate among the remaining hypotheses with the Omega Point method was 3.0% (6/200; 95% CI [0.6, 5.9]) in the initial generation and 1.3% (3/235; 95% CI [0.0, 2.9]) in the repeat generation; with the multistep pipeline it was 11.5% (23/200; 95% CI [4.6, 20.0]) and 15.2% (32/210; 95% CI [9.4, 21.4]). In tasks where both sets were retained, the match rate with the Omega Point method minus that with single-request generation was −16.5 percentage points (95% CI [−23.0, −10.5]; 40 tasks) in the initial generation and −17.8 percentage points (95% CI [−23.6, −12.4]; 45 tasks) in the repeat generation.

Leaving aside the initial Opus generations, the overall match rate of the other three models ranged from 13.2% to 16.8% with a single request, from 16.0% to 18.8% with the multistep pipeline, and from 2.8% to 7.6% with the Omega Point method. The difference between the multistep pipeline and single-request generation had p > 0.05 for all three models after Holm correction across nine comparisons.

M.2. Degree of cueing and generation

In the 38 tasks with a cueing score of 0 or 1, the overall match rate in single-request generation was 10.5% (20/190) for Astra, 13.2% (25/190) for Grok, 14.7% (28/190) for Opus, and 11.1% (21/190) for Gemini, against 14.4%, 16.8%, 18.0%, and 13.2% across all 50 tasks. In the Opus repeat generation, it was 13.7% (26/190) against 18.0%.

Under the prespecified alternative coding of borderline research questions, the correlation between the cueing score and the match rate was ρ = −0.06 (95% CI [−0.34, 0.23]; uncorrected p = 0.703). For the joint cueing score of the research question and the background, the correlation with the match rate at the primary threshold was ρ = 0.29 (95% CI [−0.06, 0.59]; uncorrected permutation p = 0.040). This exploratory finding, the between-group difference, and the recalculation on the 38 tasks are consistent with cueing in the prompt contributing to matches with the reference finding.

M.3. Comparison of generators on shared tasks

The comparison uses the 48 tasks common to the three sets, 240 hypotheses per generator. On these tasks, the final selected list contains 14 matching hypotheses of 19 for Grok, 9 of 19 for Gemini, and 13 of 20 for Opus. Two tasks in the full Gemini and Opus sets are excluded here, so the counts can differ from Table 6.

Generators compared, first vs second▲Match rate among selected hypotheses, % (n/N)▲Difference in match rates, pp [95% CI]▲Proportion of matching hypotheses found, % (n/N)▲Difference in sensitivity, pp [95% CI]▲
Grok 4.5 vs Gemini 4 Argon73.7% (14/19) vs 47.4% (9/19)26.3 [−0.2, 52.6]35.0% (14/40) vs 28.1% (9/32)6.9 [−9.7, 24.0]
Grok 4.5 vs Claude Opus 5.573.7% (14/19) vs 65.0% (13/20)8.7 [−21.2, 37.3]35.0% (14/40) vs 29.5% (13/44)5.5 [−11.2, 21.2]
Gemini 4 Argon vs Claude Opus 5.547.4% (9/19) vs 65.0% (13/20)−17.6 [−46.4, 11.0]28.1% (9/32) vs 29.5% (13/44)−1.4 [−21.8, 18.4]
Claude Opus 5.5, initial vs repeat generation61.9% (13/21) vs 58.8% (10/17)3.1 [−24.8, 31.3]29.5% (13/44) vs 23.3% (10/43)6.3 [−7.6, 19.6]

Each difference is the first value minus the second. CIs come from 10,000 paired resamples of the 48 tasks (for the last row, of the 49 tasks complete in both Opus sets). Every difference interval includes zero, so the comparison identifies no generator as better under this rule.

The random answers for the Opus repeat generation were recorded on 28 October at 22:44 UTC, before the hypothesis set was frozen at 22:45 and before the first request to the predictors at 22:45:57. All 213 requests to the four models passed the check that tools were disabled. For one task, Grok twice failed to return a forecast, and the task’s five hypotheses were excluded as a technical failure. At the strict threshold, 2 of the 17 selected hypotheses matched the reference finding in the repeat generation, as did 2 of the 22 selected in the initial generation.

M.4. Number of forecasts

The unit is one forecast by one model for one hypothesis. Each of the four models forecast every hypothesis in the set, receiving the five hypotheses of one task per request.

Generator▲Hypotheses in the set▲Forecasts by the four models▲
Grok 4.5240960
Gemini 4 Argon2501000
Claude Opus 5.52501000

M.5. Final selection under the two match thresholds

The selected lists are unchanged. The denominator of the match rate among selected hypotheses is the number of selected hypotheses; the denominator of sensitivity is the number of matching hypotheses in the full set at the same threshold. CIs come from 10,000 resamples of whole tasks.

Generator▲Score from a majority of judges▲Match rate among selected hypotheses, % (n/N) [95% CI]▲Selection sensitivity, % (n/N) [95% CI]▲
Grok 4.5at least 373.7% (14/19) [52.6, 93.3]35.0% (14/40) [25.0, 46.2]
Grok 4.5at least 426.3% (5/19) [7.1, 47.4]71.4% (5/7) [33.3, 100.0]
Gemini 4 Argonat least 347.4% (9/19) [25.0, 70.6]27.3% (9/33) [13.6, 42.9]
Gemini 4 Argonat least 431.6% (6/19) [11.1, 53.8]66.7% (6/9) [37.5, 100.0]
Claude Opus 5.5at least 363.6% (14/22) [42.9, 82.4]31.1% (14/45) [18.4, 43.9]
Claude Opus 5.5at least 49.1% (2/22) [0.0, 21.1]40.0% (2/5) [0.0, 100.0]
Claude Opus 5.5, repeat generationat least 358.8% (10/17) [35.0, 81.8]23.3% (10/43) [12.5, 34.7]
Claude Opus 5.5, repeat generationat least 411.8% (2/17) [0.0, 28.6]66.7% (2/3) [0.0, 100.0]

M.6. Predictor panel recommendations and a random list of the same length

For the predictor panel, the table gives the observed result; for random selection, it gives the mean and the central 95% range of the number of matching hypotheses under the exact distribution (Appendix L.3). Because the distribution is discrete, the range can cover more than 95% of possible outcomes. Table 6 gives confidence intervals for the panel result.

Generator▲Predictor panel, matching / selected▲Random selection, mean number matching▲Random selection, central 95% range▲Full set, matching / total▲
Grok 4.514/19 (73.7%)3.17 of 19 (16.7%)0 to 6 of 19 (0.0% to 31.6%)40/240 (16.7%)
Gemini 4 Argon9/19 (47.4%)2.51 of 19 (13.2%)0 to 6 of 19 (0.0% to 31.6%)33/250 (13.2%)
Claude Opus 5.514/22 (63.6%)3.96 of 22 (18.0%)1 to 8 of 22 (4.5% to 36.4%)45/250 (18.0%)

When a single model’s positive forecasts on the Grok hypotheses are randomly subsampled to 19, the upper bounds of the central ranges are 12 for Sonnet, Astra, and Gemini and 9 for Grok, whereas the predictor panel selected 14 matching hypotheses. On the Gemini hypotheses, the panel’s 9 of 19 lies within the range of 3 to 10 obtained by subsampling Gemini’s own 53 “yes” forecasts to 19 (18 of the 53 match). On the Opus hypotheses, the panel’s 14 of 22 exceeds by one the upper bound of the range of 6 to 13 obtained by subsampling Astra’s 49 “yes” forecasts to 22 (21 of the 49 match).

M.7. Robustness checks for the final selection

Of the 38 tasks with a cueing score of 0 or 1, the match rate among selected hypotheses was calculated on 36 tasks for Grok, 38 for Gemini and Opus, and 37 for the Opus repeat generation; the 95% CIs were [33.3, 90.9], [11.1, 62.5], [38.5, 84.2], and [22.2, 85.7]. In the full sets for the same tasks, 12.8% (23/180), 11.1% (21/190), 14.7% (28/190), and 13.0% (24/185) of hypotheses matched the reference finding.

With judges left out one at a time, the largest drop was for the Opus hypotheses without the xAI judge: from 63.6% to 54.5% (12/22). Leaving out the judge from Google, the developer of the Gemini predictor, did not change the result for the Grok hypotheses and removed one match among the selected Opus hypotheses in both generations. The ratio of the match rate among selected hypotheses to the baseline match rate ranged from 3.5 to 4.8. With two judges left, the match rule is stricter, so part of the drop reflects the change of rule.

Without the predictor from the generator’s own developer, the 95% CIs for the match rate among selected hypotheses were [47.6, 86.7] for Grok, [23.1, 66.7] for Gemini, [35.0, 70.8] for Opus, and [26.1, 65.6] for the Opus repeat generation. The proportion of matching hypotheses found rose by 0.0 to 6.9 percentage points. Astra stayed in the panel in every set, because its developer built none of the three generators. In the repeat forecast run, the CIs for the match rate among selected hypotheses were [46.7, 88.9] for Grok, [26.7, 75.0] for Gemini, and [43.5, 86.4] for Opus; 235, 245, and 240 hypotheses received forecasts in both runs. The odds ratio per one-point increase in clarity score was 2.02 (95% CI [1.04, 3.95]; p < 0.05), and adding feasibility to the regression left the estimates almost unchanged. The mean originality score was 4.86 for selected and 5.08 for excluded hypotheses; one Opus task had no excluded hypothesis left, so the comparison covers 59 pairs. By the lexical criterion, matching hypotheses named on average 13.0% of key elements and the remaining hypotheses 4.1%.

M.8. The “effect in the favourable direction” rule on the 50/50 set

Among the 61 labelled statements, 35 of the 38 confirmed and 18 of the 23 refuted statements had a favourable direction. Model forecasts agreed with the rule on 50.8% to 62.3% of labelled statements. On the 39 unlabelled statements, the models were correct in 61.5% to 82.1% of cases.

Appendix N. Contents of the published data§

Reports of the forecasting runs of 22 to 24 October are published at https://eternalsearch.net/omega/<directory>/report.json, with the logs and saved requests in the same directories: experimental-support-20261022, direct-confirmation-20261022, direct-confirmation-grok-20261022, balanced-confirmation-20261022, balanced-opus-20261023, grok-generated-confirmation-20261023, gemini-generated-confirmation-20261023, opus-generated-confirmation-20261023, and selection-replication-20261023. The final selected lists are in the selected.csv files in the two-stage-unanimous-20261023, two-stage-unanimous-gemini-20261023, and two-stage-unanimous-opus-20261023 directories.

Forecasts and selection results for the hypotheses of the Opus repeat generation are in the opus-isolated-confirmation-20261028, two-stage-unanimous-opus-isolated-20261028, and opus-isolation-selection-20261028 directories. The repeat forecast run, with verification of each request’s isolation, is in forecast-repeat-20261030-v2/comparison.json, and the originality scores of selected and excluded hypotheses are in the selection-originality-20261030 directory. The Opus repeat-generation logs verifying each request’s isolation, the records of Claude refusals with full model responses, the reproduction of the earlier configuration, and the recalculations on the 38 tasks and without each developer’s judge are available on request.

The data description sets out the structure of the row-level data and the difference between the outcome y and the decision selected. In analysis.json, the selected field is computed from the four actual forecasts. The unused yes field, which wrongly contained false in the historical reports, has been removed from the row-level data of this edition; the archived reports are kept in their original form. The data release keeps the checksums of the judge scores.

Appendix O. Control rules and robustness checks§

These analyses use the published files and the saved model responses; three of them (O.7, O.9, and O.14) also used new model responses. All were added after the results were known. Intervals are 95% percentile intervals over resamples of tasks (of articles in O.11).

O.1. Matches by position in the generator’s response

Generator▲Position 1▲Position 2▲Position 3▲Position 4▲Position 5▲
GPT-6 Astra36.0% (18/50)16.0% (8/50)10.0% (5/50)8.0% (4/50)2.0% (1/50)
Grok 4.536.0% (18/50)20.0% (10/50)8.0% (4/50)12.0% (6/50)8.0% (4/50)
Claude Opus 5.538.0% (19/50)20.0% (10/50)12.0% (6/50)8.0% (4/50)12.0% (6/50)
Gemini 4 Argon40.0% (20/50)12.0% (6/50)4.0% (2/50)10.0% (5/50)0.0% (0/50)
Four generators37.5% (75/200)17.0% (34/200)8.5% (17/200)9.5% (19/200)5.5% (11/200)
Four generators, strict threshold10.0% (20/200)1.0% (2/200)0.5% (1/200)0.5% (1/200)0.5% (1/200)

Single request, 50 tasks. In 75 of the 112 generator-task pairs with at least one match, the first-listed hypothesis matched; allocating the matches to positions at random within each pair gives an expected 31.2 (exact test, p < 0.001). Judges saw the five hypotheses of a set in a pseudo-random order fixed for the task and generation method; the proportion of votes of 3 or higher by displayed position was 16.8% (101/600), 18.3% (110/600), 13.7% (82/600), 14.0% (84/600), and 18.5% (111/600) for positions 1 to 5 (the three judges from other developers). The first-listed hypothesis was displayed first in 60 of 200 sets.

O.2. Convergence of hypotheses across generators

Generators in the group▲Hypotheses▲Match rate [95% CI]▲
13307.6% (25/330) [4.8, 10.7]
2786.4% (5/78) [0.0, 13.8]
35211.5% (6/52) [2.2, 22.2]
454022.2% (120/540) [15.9, 29.0]

Single request, 1000 hypotheses. Within each task, groups are connected components of hypotheses whose statement embeddings (embeddinggemma 300m, 768 dimensions) have a cosine similarity of at least 0.75, computed across the four generators of this study. The match rate of groups shared by all four generators exceeded that of hypotheses without a counterpart by 14.6 pp [8.2, 21.3]. The mean number of generators in a hypothesis’s group was 3.53, 2.94, 2.74, 2.52, and 2.28 for positions 1 to 5. Per task, the number of generators with at least one matching hypothesis was none in 10 tasks, one in 7, two in 12, three in 3, and four in 18.

O.3. The first-listed hypothesis as a control rule

Set▲Unanimity, PPV▲First-listed, PPV [95% CI]▲PPV difference, pp [95% CI]▲Sensitivity difference, pp [95% CI]▲
Grok 4.573.7% (14/19)37.5% (18/48) [25.0, 52.1]36.2 [19.2, 54.5]−10.0 [−25.6, 2.9]
Gemini 4 Argon47.4% (9/19)40.0% (20/50) [26.0, 54.0]7.4 [−12.7, 28.0]−33.3 [−52.6, −15.4]
Claude Opus 5.563.6% (14/22)38.0% (19/50) [24.0, 52.0]25.6 [9.7, 43.0]−11.1 [−24.4, 1.8]
Three sets61.7% (37/60)38.5% (57/148) [27.2, 50.0]23.2 [13.0, 32.6]−16.9 [−27.5, −7.8]
Claude Opus 5.5, repeat generation58.8% (10/17)30.6% (15/49) [18.4, 42.9]28.2 [9.2, 48.6]−11.6
Model▲OR of selection [95% CI]▲OR of first position [95% CI]▲
Selection9.01 [4.70, 21.21]—
Selection and first position3.30 [1.54, 8.06]4.08 [2.09, 8.52]

Differences are unanimity minus first-listed hypotheses on the same resamples. Selected hypotheses by position in the three sets: 50, 7, 1, 2, and 0 for positions 1 to 5; 6 of the 61 matching hypotheses in positions 2 to 5 were selected. The second table is a conditional logistic regression with each generator’s tasks as strata (147 strata, 85 of them informative). The three generators answered the same tasks, so the resampling unit is the task with all of its strata; intervals come from 2000 resamples of tasks.

O.4. Selection-adjusted estimate for the procedure that picks a rule

Rule (members, minimum number of “yes”)▲List length▲Matches▲PPV, %▲Sensitivity, %▲
Sonnet, Astra, Gemini, Grok, Opus ≥ 5161275.030.0
Sonnet, Astra, Gemini, Grok ≥ 4191473.735.0
Sonnet, Astra, Grok, Opus ≥ 4181372.232.5
Sonnet, Astra, Grok ≥ 3231669.640.0
Sonnet, Astra, Gemini, Opus ≥ 4191368.432.5
Sonnet, Astra, Gemini ≥ 3221568.237.5
Criterion for choosing a variant▲Apparent PPV of the best variant, %▲Leave-one-task-out PPV▲Optimism-corrected PPV, %▲
highest PPV, list of at least 1075.066.7% (12/18)69.7
highest PPV, sensitivity of at least 30%75.066.7% (12/18)69.8
highest F156.147.7% (21/44)50.9

Grok hypotheses; 112 variants yield 87 distinct lists. The chosen rule (unanimity of Sonnet, Astra, Gemini, and Grok) ranks 2nd by PPV among variants with lists of at least 10, after unanimity of all five models. The three right-hand columns describe the procedure “take the variant that this criterion ranks first”, not the chosen rule: in 46 of the 48 folds the criterion took unanimity of all five models, and in 1 fold it took the chosen rule. Leave one task out: for each of the 48 tasks, the variant is chosen by the criterion on the other 47 tasks and applied to the held-out task. Optimism correction: the mean difference between apparent and out-of-sample PPV over 2000 bootstrap resamples, with the selection repeated in each. For the chosen rule itself, the check is its application to the Gemini and Opus hypotheses, fixed on 23 October before those forecasts were obtained (Section 3.5 and Appendix L.4).

O.5. Random subsets of the best single model and ranking by votes

Set▲Unanimity list▲Matches▲Best single model▲Its “yes” forecasts▲Matches among them▲Expected in a random subset▲p of at least as many▲
Grok 4.51914GPT-6 Astra53258.960.0043
Gemini 4 Argon199Gemini 4 Argon53186.450.1084
Claude Opus 5.52214GPT-6 Astra49219.430.0087
Claude Opus 5.5, repeat generation1710Gemini 4 Argon61215.850.0152
List length▲By votes▲By votes, then listing order▲By listing order▲Random list▲
3018.518.611.64.8
6037.037.023.19.6
7541.942.728.912.0
10953.053.042.017.4
14859.661.557.023.6
15060.061.957.323.9
30080.481.282.347.8

Three sets together, 740 hypotheses with 118 matches; cells give the expected number of matches in a list of the given length, with ties broken at random. The combined unanimity lists have length 60, and the first-listed hypotheses number 148.

Set▲All▲At least 1 “yes”▲At least 2▲At least 3▲All four▲
Grok 4.5240 / 40132 / 3773 / 3037 / 1919 / 14
Gemini 4 Argon250 / 33130 / 2272 / 1833 / 1519 / 9
Claude Opus 5.5250 / 45134 / 3270 / 2339 / 1922 / 14
Three sets740 / 118396 / 91215 / 71109 / 5360 / 37

Each cell gives hypotheses in the list / matching hypotheses (Figure 3).

O.6. Judge panels, leniency, and self-preference

Judge▲Votes ≥ 3, %▲Votes ≥ 4, %▲Mean score▲Generator from its developer▲Difference in differences, points [95% CI]▲Same for votes ≥ 3, pp [95% CI]▲
GPT-5.6 Sol16.02.21.99GPT-6 Astra0.13 [0.07, 0.18]0.6 [−1.9, 3.3]
Grok 4.712.91.52.04Grok 4.50.00 [−0.04, 0.05]2.2 [0.6, 4.0]
Claude Opus 5.512.14.11.32Claude Opus 5.50.03 [−0.03, 0.10]0.3 [−1.3, 2.1]
Gemini 4 Argon (medium)11.72.01.66Gemini 4 Argon0.15 [0.10, 0.20]0.9 [−1.4, 3.2]

All 2997 hypotheses. Difference in differences: the judge’s score minus the other judges’ scores on hypotheses from its own developer’s generator, compared with the same difference on the other generators’ hypotheses.

Judges deciding the outcome▲GPT-6 Astra▲Grok 4.5▲Claude Opus 5.5▲Gemini 4 Argon▲
Primary rule (judge from the generator’s developer excluded)36424533
Sol, Grok 4.7, Opus38474433
Sol, Grok 4.7, Gemini36424535
Sol, Opus, Gemini38424129
Grok 4.7, Opus, Gemini36454231
All four judges, at least 2 votes39474835
All four judges, at least 3 votes35413829

Matching hypotheses out of 250 in single-request generation.

O.7. Composition of the predictor panel

Panel▲Grok 4.5▲Gemini 4 Argon▲Claude Opus 5.5▲Claude Opus 5.5, repeat generation▲
Four models (primary rule)73.7% (14/19)47.4% (9/19)63.6% (14/22)58.8% (10/17)
Without Claude Sonnet 5.561.5% (16/26)50.0% (14/28)53.6% (15/28)46.4% (13/28)
Without GPT-6 Astra60.9% (14/23)43.5% (10/23)57.1% (16/28)50.0% (10/20)
Without Gemini 4 Argon69.6% (16/23)45.0% (9/20)60.0% (15/25)43.5% (10/23)
Without Grok 4.568.2% (15/22)47.4% (9/19)62.5% (15/24)55.0% (11/20)
Gemini 3.8 Flash in place of Gemini 4 Argon73.3% (11/15)50.0% (8/16)57.9% (11/19)46.7% (7/15)

Cells give the PPV of the list selected unanimously by the panel. The predictor from the generator’s developer is Grok 4.5 for the Grok hypotheses, Gemini 4 Argon for the Gemini hypotheses, and Claude Sonnet 5.5 for the Opus hypotheses. Gemini 3.8 Flash forecasts were obtained under a protocol fixed before the first request; this model was not a judge in this study.

O.8. Dependence of predictor errors

Set▲Unanimous “yes” for non-matching hypotheses▲Expected under independence▲Ratio [95% CI]▲PPV under independence, %▲Observed PPV▲
Grok 4.550.2024.9 [6.3, 59.7]96.773.7% (14/19)
Gemini 4 Argon100.3628.1 [15.4, 50.6]82.647.4% (9/19)
Claude Opus 5.580.2927.8 [13.9, 52.5]89.763.6% (14/22)
Three sets230.8427.5 [18.3, 40.7]91.761.7% (37/60)
Models required to say “yes”▲Combinations▲Mean list length▲Mean PPV, %▲Mean sensitivity, %▲
14195.034.753.4
26102.247.740.7
3472.256.934.7
4160.061.731.4

Independence given the outcome: the expected number of unanimous “yes” forecasts is the product of the four models’ “yes” rates among non-matching (or matching) hypotheses, multiplied by the number of such hypotheses. Second table: unanimity within every combination of the given number of the four models, three sets together.

O.9. Reference findings extracted by Gemini 4 Argon and re-scoring

Set▲Tasks▲Matches, Sol / Gemini reference▲Unanimity PPV, Sol / Gemini reference▲Gain over first-listed, pp [95% CI], Sol / Gemini▲
Grok 4.54537 / 3276.5% (13/17) / 70.6% (12/17)38.7 [19.6, 57.8] / 37.3 [17.8, 57.8]
Gemini 4 Argon4729 / 3444.4% (8/18) / 44.4% (8/18)4.0 [−16.9, 25.3] / 6.1 [−13.4, 26.1]
Claude Opus 5.54744 / 4063.6% (14/22) / 63.6% (14/22)25.3 [9.6, 42.1] / 29.6 [9.9, 48.1]
Three sets139110 / 10661.4% (35/57) / 59.6% (34/57)22.6 [11.8, 33.6] / 24.4 [13.2, 36.0]

Gemini 4 Argon at high reasoning effort received the original extraction request. It accepted 47 of the 50 tasks, judging R42642518, R42647635, and R42696595 unsuitable, and for all 47 Grok 4.7 judged the core finding, the mechanism or target, and the direction of the effect to be the same as in GPT-5.6 Sol’s version. The three judges from other developers re-scored the single-request hypotheses of the three selection sets against Gemini’s reference findings with the original request, order, and settings; the original request, rebuilt by the same script, matched the logged request byte for byte. Votes of 3 or higher against the new and the original reference findings agreed in 95.4% of cases (1989/2085): GPT-5.6 Sol 94.4% (656/695), Grok 4.7 96.6% (454/470), Claude Opus 5.5 94.6% (435/460), and Gemini 4 Argon (medium) 96.5% (444/460). Outcomes agreed in 95.7% (665/695). The Sol-reference values are recalculated on the same tasks.

O.10. Calibration of the probability of confirmation

Predictor▲Hypotheses▲Mean probability▲Match rate▲Brier score▲Skill score [95% CI]▲Calibration slope▲Match rate at probability 0–0.2▲0.2–0.4▲0.4–0.6▲0.6–1.0▲
Claude Sonnet 5.52500.240.140.1230.01 [−0.21, 0.13]1.146.3% (5/79)12.9% (17/132)35.3% (12/34)40.0% (2/5)
Claude Opus 5.52150.410.140.187−0.60 [−1.54, −0.17]1.090.0% (0/10)6.5% (6/93)17.5% (14/80)28.1% (9/32)
Claude Haiku 5.52500.260.140.135−0.10 [−0.33, 0.03]0.7710.8% (7/65)13.5% (21/155)23.1% (6/26)50.0% (2/4)
Claude Opus 4.82400.430.140.197−0.62 [−1.34, −0.24]1.460.0% (0/2)7.5% (6/80)15.6% (22/141)35.3% (6/17)
GPT-6 Astra2450.470.140.228−0.86 [−1.72, −0.39]0.9920.0% (1/5)5.5% (4/73)12.0% (14/117)32.0% (16/50)
Gemini 4 Argon2500.510.140.250−1.03 [−2.03, −0.48]1.150.0% (0/7)6.8% (5/74)7.6% (6/79)27.8% (25/90)
Grok 4.52250.400.140.180−0.52 [−1.19, −0.18]1.250.0% (0/5)6.2% (7/112)19.1% (17/89)36.8% (7/19)

The 250 GPT-6 Astra single-request hypotheses with a numerical probability under the detailed prompt. Skill score: 1 minus the ratio of the Brier score to that of a constant forecast equal to the match rate; negative values mean a larger error than the constant forecast. Slope: logistic regression of a match on the logit of the probability. The probability refers to experimental confirmation, whereas the outcome counts a correct hypothesis about a mechanism other than the article’s as a non-match.

O.11. The 50/50 set by publication date

Model▲Stated training cutoff▲Balanced accuracy, 25 articles before 1 September, %▲Balanced accuracy, 25 articles from 1 September, %▲Difference, pp [95% CI]▲
Claude Sonnet 5.52026-06-3072.076.04.0 [−12.0, 20.0]
GPT-6 Astra2026-04-3076.066.0−10.0 [−26.0, 6.0]
Gemini 4 Argon2026-03-3182.074.0−8.0 [−24.0, 8.0]
Grok 4.52026-01-3180.070.0−10.0 [−28.0, 8.0]
Claude Opus 5.52026-06-3078.068.0−10.0 [−24.0, 6.0]
Five-model unanimity—74.066.0−8.0 [−22.0, 6.0]

Mean change for Opus and Sonnet minus mean change for Astra, Gemini, and Grok: 6.3 pp [−5.0, 18.0]. If training-data leakage contributed to performance on the earlier articles, this value would be negative. Intervals come from 2000 resamples of articles within each period.

O.12. Second random sequence for the Astra hypotheses

Threshold▲Match rate▲PPV▲NPV▲Sensitivity▲Specificity▲PPV range across fair coins▲
Primary threshold14.4% (36/250)13.0% (17/131)84.0% (100/119)47.2% (17/36)46.7% (100/214)[10.0, 18.8]
Strict threshold1.6% (4/250)0.8% (1/131)97.5% (116/119)25.0% (1/4)47.2% (116/246)[0.0, 3.2]

Seed 20261104 was fixed in a protocol file committed before the calculation; the sequence has 131 “yes” answers among 250. The range covers the central 95% of 10,000 further fair-coin sequences on the same hypotheses.

O.13. Selection adjusted for lexical overlap, length, mechanisms, and entities

Model▲Unanimous selection▲Named key elements, per 10 pp▲Length, per 100 words▲Named mechanisms, per one▲Named entities, per one▲
Selection9.01 [4.70, 21.21]————
Selection and key elements7.55 [3.98, 17.86]1.90 [1.53, 2.86]———
All covariates5.47 [2.75, 13.14]1.84 [1.47, 2.86]13.51 [1.17, 253.12]0.85 [0.39, 1.63]1.01 [0.91, 1.13]

Odds ratios [95% CI] from conditional logistic regression on the three sets, with each generator’s tasks as strata and the task as the resampling unit. Named key elements follow the rule of Appendix K.8. Gemini 4 Argon counted mechanisms and entities in the 1000 single-request hypotheses. Selected versus other hypotheses: mean share of named key elements 0.130 and 0.051, mean length 140.5 and 133.1 words, mechanisms 1.20 and 1.35, and entities 12.60 and 12.77.

O.14. Blind re-coding of the question cue

Codings▲Same score▲Cohen’s kappa▲Quadratic-weighted kappa▲
Original and Gemini 4 Argon39/500.590.71
Original and Grok 4.734/500.470.65
Gemini 4 Argon and Grok 4.743/500.750.81
Coding▲Questions with score 2▲Match rate, score 2▲Match rate, scores 0–1▲Difference, pp [95% CI]▲Spearman’s rho [95% CI]▲
Original1225.8% (62/240)12.4% (94/760)13.5 [2.1, 25.2]0.17 [−0.14, 0.45]
Gemini 4 Argon535.0% (35/100)13.4% (121/900)21.6 [−0.9, 38.0]0.18 [−0.17, 0.47]
Grok 4.7637.5% (45/120)12.6% (111/880)24.9 [5.5, 38.8]0.23 [−0.10, 0.51]
Score 2 from both re-coders535.0% (35/100)13.4% (121/900)21.6 [−0.9, 38.0]0.30 [−0.11, 0.57]

Single request, four generators. The re-coders received the frozen rubric, the background, the question, the reference finding, and its key elements, but no outcome. Intervals come from 10,000 resamples of tasks (2000 for rho); the intervals of the original coding therefore differ slightly from those of the primary analysis in Section 3.1.

O.15. Difference in the gain from selection between generators

Generators compared▲Shared tasks▲Gain of each, pp▲Difference, pp [95% CI]▲p▲Holm-adjusted p▲
Grok 4.5 and Gemini 4 Argon4836.2 and 7.828.4 [1.4, 56.1]0.0530.16
Claude Opus 5.5 and Gemini 4 Argon5025.6 and 7.418.3 [−7.8, 44.6]0.170.34
Grok 4.5 and Claude Opus 5.54836.2 and 27.58.7 [−18.5, 34.6]0.540.54

The gain is the match rate among selected hypotheses minus the match rate among first-listed hypotheses of the same set, computed on the tasks the two generators share. Under the null hypothesis, the two generators’ whole hypothesis sets exchange labels within each task, the procedure that compares generation methods in Section 2.10; 19,999 permutations, with Holm correction across the three pairs. Intervals come from 10,000 paired resamples of the shared tasks.

O.16. Selection on the tasks whose reference findings the manual audit confirmed

Tasks kept▲Number of tasks▲Unanimity, PPV▲First-listed, PPV▲Sensitivity▲Gain, pp [95% CI]▲
All tasks5061.7% (37/60)38.5% (57/148)31.4% (37/118)23.2 [13.0, 32.6]
Reference finding scored 3 or not audited3863.0% (29/46)38.4% (43/112)33.7% (29/86)24.7 [13.0, 35.7]
Reference finding scored 1 or 21257.1% (8/14)38.9% (14/36)25.0% (8/32)18.3 [0.0, 35.1]

Three sets together. Of the 20 audited reference findings, 8 scored 3, 11 scored 2, and 1 scored 1 on the accuracy scale of Appendix P.3; the reference findings of the remaining 30 tasks were not in the frozen sample. The middle row therefore keeps the tasks whose reference finding the audit confirmed in full together with the tasks it did not cover.

O.17. Scores behind the matches of the selected lists

Set▲Selected▲Matches▲No score above 3▲Matches at the strict threshold▲Majority of two votes▲
Grok 4.51914451
Gemini 4 Argon199160
Claude Opus 5.522141122
Three sets603716133

Scores of the three judges from developers other than the generator’s. A match with no score above 3 agrees with the mechanism or target of the reference finding while at least one of its key elements is missing or replaced, the level the primary threshold accepts (Section 2.4). The last column counts the matches whose majority one changed vote would reverse.

Appendix P. Manual audit of hypotheses and reference findings§

P.1. Frozen samples and scoring

On 5 November 2026, we completed the audit of both frozen samples; all scores were given by one of us. The hypothesis sample contains 100 of the 1000 single-request hypotheses from the four generators, drawn from 44 of the 50 tasks. The rater saw the background, question, reference finding, key elements and hypothesis, and scored agreement with the reference finding on the judges’ 0 to 5 scale. Generator identity, original position, judge scores, selection status and sampling stratum were hidden. The sample was frozen on 2 November, before the first score (SHA-256 2c2c1cf5fc1c820c4cdbf9b5c7d27ed1b2dbb53f8dd602899b1acfea44c8f18e).

The table gives stratum sizes and results at the primary threshold. “Unselected” means not on the 60-item list of the three selection sets. The 40 split-vote records are unselected records sampled specifically for a split vote. Population estimates use inverse-inclusion weights: the population size of the stratum divided by its sample size.

Stratum▲Population▲Sample▲Matches, manual audit▲Matches, judges▲Same binary decision▲
Selected by unanimity603019/3020/3025/30
Unselected, two positive judge votes362015/2020/2015/20
Unselected, one positive judge vote56204/200/2016/20
Unselected, three positive judge votes831511/1515/1511/15
Unselected, zero positive judge votes765151/150/1514/15
Total100010050/10055/10081/100

The reference sample comprises the first 20 of the 50 tasks ordered by SHA-256 of benchmark-reference-check-v1: followed by the task identifier. It was frozen on 4 November, before the first check (SHA-256 05f6a81400aae45ad2c5bc8b9b5f25a669c3bb032701a630fa315ce553ca69eb). We checked 16 reference findings against full texts; reference findings nos. 1 (R42601407), 4 (R42821663), 11 (R42759510) and 20 (R42822442) were checked against abstracts, and their score of 3 means agreement with those abstracts. The scale distinguishes a correct finding with all key elements (3), a correct central finding with an imperfect key element (2), a substantive discrepancy in the central finding (1), and an incorrect reference (0).

P.2. Agreement with the judges

The comparator is the majority decision of the three judges from other developers, which is equivalent to thresholding their median score. At the primary threshold, both we and the judges scored 43 hypotheses as matches and 38 as non-matches; the judges alone scored 12 as matches, and we alone scored 7. At the strict threshold, the corresponding counts were 8, 84, 2, and 6.

Threshold▲Agreement [95% CI], %▲Cohen’s kappa [95% CI]▲Matches, manual audit / judges▲
Primary, score ≥ 381.0 [73.4, 88.2]0.62 [0.46, 0.76]50/100 / 55/100
Strict, score ≥ 492.0 [86.9, 96.6]0.62 [0.24, 0.84]14/100 / 10/100

The manual score equalled the median judge score in 62/100 cases and differed from it by at most one point in 99/100; quadratic-weighted kappa was 0.73. Reweighting the five strata to their population sizes gave an estimated agreement at the primary threshold of 89.7% [77.1, 97.0] and a kappa of 0.64 [0.37, 0.87]. These are weighted estimates; the unweighted percentages describe the enriched validation sample.

Among the 30 sampled selected hypotheses, 19 matched by the manual score (63.3%; 95% CI [41.7, 81.8]) and 20 by the judges’ decision (66.7%); the paired difference was −3.3 pp [−19.0, 10.7]. At the strict threshold, 11/30 matched by the manual score and 8/30 by the judges’ decision. Intervals come from 10,000 bootstrap resamples of the 44 sampled tasks, keeping each task’s hypotheses together and the original sampling weights fixed (seed 20261105). Agreement with the manual audit checks the judges’ decisions on matching the reference finding; the outcome is still defined as correspondence with the published finding.

P.3. Accuracy of reference findings

Across the reviewed sources, eight reference findings scored 3 (40.0%, 8/20), eleven scored 2 (55.0%, 11/20), and one scored 1 (5.0%, 1/20); the central finding was rated correct in 15/16 full-text checks and 4/4 abstract checks. Four of the eight reference findings scoring 3 were checked against abstracts. The one scoring 1 was no. 9, R42844664, on time-restricted feeding, microbial acetate and cardiovascular ageing (doi:10.1111/acel.70728). The primary analysis keeps the original reference findings and outcomes; excluding that whole task is a sensitivity analysis.

Source reviewed▲Reference findings▲Score 3▲Score 2▲Score 1▲Score 0▲
Full text1641110
Abstract44000
Total2081110

Section 3.8 of the article explains how the four abstract-only checks are interpreted; our explanation of the score of 1 for R42844664 follows.

A score of 1 was assigned to reference finding no. 9 (R42844664, Aging Cell), which presents the sequence “acetate — GPR43 — HDAC3 inhibition — H4K8 acetylation — suppression of NLRP3-dependent pyroptosis” as an established mechanism. For GPR43 and HDAC3, the study reports expression levels only (Figs. 6O and S14); the study’s authors performed no loss-of-function experiments using gene knockout, receptor antagonists, or enzyme inhibitors, and the acetate-associated change in HDAC3 in the HUVEC experiments is reported in opposite directions in two places in the article (Fig. S11). The link between H4K8 acetylation and NLRP3 remains correlational, because chromatin immunoprecipitation at the NLRP3 promoter was not performed; the authors acknowledge in the discussion that this experiment is needed.

For the microbiota as a whole, the description “necessary and sufficient” is supported: antibiotic treatment abolishes protection, and faecal microbiota transfer reproduces it. For acetate, only sufficiency is shown (sodium acetate gavage, Figs. 6A–P), because neither acetate blockade nor an acetate-deficient donor was used. Butyric, valeric, and isobutyric acids also rose significantly together with acetate, so the data do not single out acetate. The between-group difference in Limosilactobacillus is significant in the metagenomic data but not in the 16S data (Fig. S13), and the bacterium was not given to the animals as a probiotic; “acetate-producing Limosilactobacillus” should therefore be read as the authors’ interpretation.

The receptor and deacetylase steps of this sequence, and the step from histone acetylation to pyroptosis, therefore lack causal support, which determined the score.

An abstract-only check establishes agreement with the abstract from which the reference finding was compiled. The source of every reference finding and the full explanation accompany the item-level data.

Without task R42844664, the selected lists contained 14 matches among 18 Grok hypotheses (77.8%), 9 among 19 Gemini hypotheses (47.4%), and 13 among 21 Opus hypotheses (61.9%); pooled, 36/58 (62.1%), against 37/60 (61.7%) across all tasks. In the manual-audit sample, agreement at the primary threshold was 81/99, and both we and the judges scored 19 of the 29 remaining selected hypotheses as matches. Item-level scores, inputs and calculation results are published together with the code of this analysis.

Table S. Selected hypotheses, reference findings and judge scores§

Supplementary material to the preprint “Benchmarking four large language models on generating and selecting hypotheses that match published findings in 50 studies of ageing biology”.

The table lists all 60 hypotheses selected for the three generators by four-model unanimity: all four predictors forecast “yes” for each of them. For each hypothesis it gives the full text, the research question of the task, the reference finding of the article and the scores of the three judges from other developers. The primary threshold requires a score of at least 3 from the majority of judges; the strict threshold requires a score of 4 or 5. Scores are listed in the order of the judges for the given generator. A majority decision at the primary threshold is called borderline when the judges split two to one: a different vote from one judge would reverse it. Borderline decisions: 6 of 60.

The texts of the hypotheses, questions and reference findings are given in full, in the language of the experiment: the models received exactly these wordings. The table was generated by code from the published selection reports; the checksums of the sources are listed in the section “Source checksums”.

No.▲Generator model▲Task▲Hypothesis▲Research question▲Full text of the hypothesis▲Reference finding of the article▲Scores of the three judges from other companies▲Match at the primary threshold▲Borderline majority decision▲Match at the strict threshold▲Article DOI▲
1Grok 4.5R42605193H4Which circulating regulator causally promotes cellular senescence and organismal ageing, and through what intracellular signaling mechanism does it act?Age-elevated circulating TGF-β1 is a causal driver of multi-tissue cellular senescence and organismal ageing. Chronic exposure of young tissues to aged plasma levels of TGF-β1 is sufficient to induce p16INK4a/p21-positive senescence and accelerate functional decline. Mechanism: Circulating TGF-β1 binds TGFBR1/2, activates Smad2/3–Smad4 complexes that transcriptionally induce CDKN2A/CDKN1A and remodel chromatin toward a stable senescence program, while also reinforcing SASP output that further elevates systemic TGF-β1. Prediction: Neutralizing circulating TGF-β1 (or tissue-specific TGFBR1 deletion) in aged animals will reduce senescence markers across distant organs and improve healthspan metrics more than neutralizing IL-6 or insulin signaling alone; conversely, sustained infusion of TGF-β1 into young animals will recapitulate the aged senescence signature even when inflammatory cytokines remain low.ANGPTL8 directly interacts with AKT2 and activates the AKT–mTOR–S6K pathway, thereby driving cell-autonomous adipocyte senescence. Deleting Angptl8 in mice reduces adipose senescence, delays age-related functional decline, and extends lifespan, while genetic or pharmacological pathway inhibition abolishes ANGPTL8-induced senescence.GPT-5.6 Sol 2; Claude Opus 5.5 1; Gemini 4 Argon 1nonono10.1111/acel.70671
2Grok 4.5R42630044H3What mechanism limits the anabolic response of aged bone to mechanical loading, and can targeted stimulation restore this response?Age-related decline in osteocyte Piezo1 (or associated ion-channel/Ca2+ entry) blunts early mechanotransduction, so downstream anabolic gene programs are not activated by physiological strain. Agonism or overexpression of Piezo1 will restore loading responsiveness in aged bone. Mechanism: Reduced Piezo1-dependent calcium influx and YAP/TAZ or NFAT activation in osteocytes cuts the initiation of load-adaptive transcriptional responses. Prediction: Aged osteocytes will show weaker load-evoked Ca2+ transients and Piezo1-dependent gene induction; Piezo1 activation will rescue formation indices after loading, while Piezo1 loss in young bone will phenocopy the aged loading defect.In aged mouse bone, activation of Piezo1 during mechanical loading promotes its membrane co-localization with connexin 43 and increases connexin 43 hemichannel activity through PI3K-Akt signaling. Hemichannel opening increases PGE2 release, suppresses sclerostin, promotes endosteal bone formation, inhibits resorption, and thereby restores the cortical anabolic response to loading.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 3yesnono10.1111/acel.70687
3Grok 4.5R42647119H1How does aging affect injury-induced neuronal reprogramming in the retina, and what mechanisms account for any age-related loss of regenerative capacity?Aging reduces injury-induced neuronal reprogramming in the retina because chronic low-grade inflammation progressively impairs Müller glial competence to activate neurogenic programs after injury. Suppressing age-associated inflammatory signaling in old retina should restore reprogramming efficiency toward youthful levels. Mechanism: Persistent innate immune activation (inflammaging) elevates NF-κB/STAT-driven glial reactivity that antagonizes Ascl1 and related neurogenic gene networks required for Müller glia-to-neuron conversion after injury. Prediction: Aged retinas will show higher baseline and post-injury inflammatory cytokine signatures coincident with fewer reprogrammed neurons; neutralizing key inflammatory mediators (e.g., IL-6/TNF or microglial depletion) will increase reprogrammed neuron yield in old but not young animals, whereas adding the same cytokines to young tissue will reduce yield.Aging suppresses Müller glia-to-neuron reprogramming by preventing activation of progenitor programs while promoting reactive and inflammatory glial states and an exaggerated retinal neuroimmune response. Dexamethasone partially restores neurogenesis, confirming that age-associated inflammation is a causal, therapeutically tractable component of the regenerative defect, alongside glial-intrinsic barriers.GPT-5.6 Sol 4; Claude Opus 5.5 4; Gemini 4 Argon 4yesnoyes10.1073/pnas.2612369123
4Grok 4.5R42647635H1Does experimentally increasing somatic mitochondrial DNA mutation burden cause premature ageing and shortened lifespan in a large mammal?Experimentally elevating the somatic mtDNA mutation rate in a large mammal will accelerate multi-tissue functional decline and shorten median and maximum lifespan relative to isogenic controls with a wild-type mtDNA polymerase proofreading capacity. Mechanism: Proofreading-deficient mtDNA polymerase increases the load of random somatic mtDNA mutations, impairing oxidative phosphorylation, elevating ROS and bioenergetic failure in post-mitotic and stem-cell niches, and thereby driving systemic ageing phenotypes. Prediction: Mutator animals will show dose-dependent earlier onset of sarcopenia, anemia, fertility loss, and cardiac dysfunction together with reduced lifespan, with these traits correlating quantitatively with measured tissue mtDNA mutation burden and respiratory-chain deficiency.Prime-edited pigs expressing proofreading-deficient POLG accumulated elevated somatic mitochondrial DNA mutations and developed systemic premature-ageing phenotypes. These included weight loss, rough hair, anaemia, tissue abnormalities, increased apoptosis, elevated senescence-associated markers, and shortened lifespan.GPT-5.6 Sol 4; Claude Opus 5.5 5; Gemini 4 Argon 4yesnoyes10.1126/sciadv.adu0632
5Grok 4.5R42665751H3How does age-associated mitochondrial dysfunction initiate and propagate inflammatory signaling that causes ovarian functional decline?Age-associated mitochondrial DNA damage in oocytes and granulosa cells releases oxidized mtDNA into the cytosol, which activates cGAS–STING and thereby initiates type-I interferon and NF-κB inflammatory programs that impair follicle survival and steroidogenesis. Mechanism: Cytosolic mtDNA sensing by cGAS–STING drives interferon and NF-κB transcription in follicular cells, creating a local inflammatory milieu that reduces oocyte competence and granulosa endocrine function. Prediction: Ovary-specific cGAS or STING deletion (or mtDNA packaging rescue) will reduce follicular inflammatory transcripts and preserve ovulation and estradiol output with age, whereas parallel inflammasome blockade alone will not fully rescue the same endpoints.Ageing-associated mitochondrial DNA leakage from oocytes activates oocyte cGAS, generating cGAMP that triggers STING signaling and passes through gap junctions to activate STING in surrounding granulosa cells. Genetic deletion of oocyte Cgas or pharmacological STING inhibition ameliorates inflammation and ovarian dysfunction, establishing this signaling axis as a causal driver of ovarian ageing.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 3yesnono10.1038/s43587-026-01195-y
6Grok 4.5R42689491H4Can in vivo correction of the HGPS-causing mutation rescue molecular, cellular, and structural defects in bone, and how does treatment timing affect efficacy?In vivo correction of the HGPS-causing LMNA mutation before a critical developmental window fully restores osteoblast differentiation, bone matrix deposition, and adult bone mechanical integrity because early progerin toxicity primarily impairs formation rather than locking in irreversible structural damage. Mechanism: Progerin-driven nuclear lamina dysfunction and DNA-damage signaling in osteoprogenitors disrupt Runx2/Osterix programs during peak bone accrual; removing progerin early reopens normal lineage progression and mineralization before cortical architecture is permanently compromised. Prediction: Correction in juvenile animals normalizes osteoblast transcriptomes, mineral apposition rate, cortical thickness, and fracture toughness to wild-type levels, whereas identical correction after skeletal maturity leaves residual deficits in geometry and strength despite molecular normalization.Adenine base editing of the pathogenic LMNA variant reduced progerin expression, restored extracellular-matrix deposition and mineralization in progeroid osteoblasts, and partially rescued bone structure and physical properties in mice. In vivo correction was strongly age-dependent, with early postnatal delivery producing substantially greater editing and skeletal benefit than later treatment.GPT-5.6 Sol 3; Claude Opus 5.5 3; Gemini 4 Argon 4yesnono10.1111/acel.70700
7Grok 4.5R42696595H3What genetic and molecular mechanism causes natural variation in seed longevity among rice varieties?Natural allelic variation in a rice IAA-amido synthetase (OsGH3-family) gene alters free auxin homeostasis in developing seeds, changing seed-coat structure and thereby storage longevity. Accessions carrying a high-activity longevity allele conjugate more IAA, produce a less permeable coat, and retain higher germination after aging. Mechanism: Auxin conjugation reduces free IAA during late seed maturation, remodeling testa cell-wall and cuticle deposition and slowing oxidative aging. Prediction: Near-isogenic lines that differ only at the OsGH3 longevity QTL will show correlated differences in free IAA, seed-coat permeability (tetrazolium uptake), and post-aging germination; CRISPR knockout of the high-longevity allele will shorten longevity without changing primary yield components when auxin is restored chemically.Natural variation at qSL6 causally determines rice seed longevity through SL6, a fatty acyl-ACP thioesterase that changes seed fatty-acid composition and increases resistance to oxidation. Desiccation-induced ABA signaling activates SL6 through VP1, while a virus-derived CT-rich element in the 5′ UTR of the ancestral SL6HHZ allele stabilizes its mRNA bidirectionally and contributes to longevity differences between indica and japonica rice.GPT-5.6 Sol 1; Claude Opus 5.5 1; Gemini 4 Argon 1nonono10.1126/sciadv.aeg8609
8Grok 4.5R42698274H4What molecular mechanism exacerbates dihydrotestosterone-induced mitochondrial dysfunction, oxidative stress and senescence during androgenetic alopecia?Dihydrotestosterone-bound androgen receptor transcriptionally represses PGC-1α in dermal papilla cells, collapsing mitochondrial biogenesis and antioxidant capacity. The resulting electron-transport inefficiency elevates ROS, which accelerates cellular senescence and shortens anagen. Mechanism: AR-dependent transcriptional downregulation of the PGC-1α–NRF1/TFAM biogenesis axis, with secondary loss of SOD2/GPX activity. Prediction: Forced PGC-1α expression or AR DNA-binding blockade will restore mtDNA copy number and oxygen consumption and reduce SA-β-gal/p16 under DHT, whereas NOX or inflammasome inhibitors will not fully rescue biogenesis markers.SFRP2 binds FSTL1 and increases its protein stability, establishing an SFRP2–FSTL1 axis that exacerbates DHT-induced mitochondrial dysfunction, oxidative stress, inflammation and senescence in dermal papilla cells. SFRP2 knockdown alleviated these defects and improved hair regeneration and follicle morphology, while FSTL1 knockdown counteracted the harmful effects of SFRP2 overexpression.GPT-5.6 Sol 2; Claude Opus 5.5 0; Gemini 4 Argon 1nonono10.1111/acel.70666
9Grok 4.5R42717261H5What mechanism induces pathogenic immune-cell senescence in ulcerative colitis, and does this senescence causally exacerbate the disease?In ulcerative colitis, chronic metabolic stress damages mitochondria in lamina propria T cells, releasing mtDNA into the cytosol; cytosolic mtDNA activates cGAS–STING, which drives a senescence program that produces a SASP that amplifies mucosal inflammation and impairs epithelial repair. Mechanism: Mitochondrial damage → cytosolic mtDNA → cGAS–STING → immune-cell senescence → SASP-driven colitis exacerbation Prediction: Selective blockade or genetic deletion of cGAS or STING in T cells will reduce senescence markers (p16, SA-β-gal), lower SASP cytokines in the mucosa, and improve histological colitis scores even when upstream mitochondrial stress persists; exogenous cytosolic mtDNA will recapitulate senescence and worsen disease in a STING-dependent manner.NAD+ metabolic dysregulation causes mitochondrial dysfunction and cytosolic mitochondrial DNA leakage, activating cGAS-STING signaling to drive CD8+ T-cell senescence. These senescent cells exacerbate colitis, while systemic senolytic treatment reduces disease severity; their associated signature also predicts severe disease and non-response to biologic therapy.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 4yesnoyes10.1111/acel.70706
10Grok 4.5R42723546H2Can a cell-free regenerative treatment alleviate UV-induced skin photoaging, and which causal intracellular mechanism mediates its protective effects?A cell-free regenerative preparation alleviates UV-induced photoaging by restoring mitophagy in keratinocytes and fibroblasts, which clears dysfunctional mitochondria and thereby reduces mitochondrial danger-signal release that drives senescence and sterile inflammation. Mechanism: Enhanced PINK1/Parkin-dependent mitophagy lowers mtDNA and ROS leakage, interrupting NLRP3 inflammasome activation and SASP maintenance. Prediction: Pharmacologic or genetic blockade of mitophagy will abolish the therapy’s anti-senescence and anti-inflammatory benefits even when barrier and collagen markers partially recover; mitophagy flux reporters and circulating mtDNA will normalize only in treated, mitophagy-competent skin.Human umbilical cord mesenchymal stem cell secretome protects against UV-induced photoaging by enhancing mitophagy, which clears damaged mitochondria and reduces cytosolic mitochondrial DNA leakage. This suppresses cGAS-STING signaling and downstream inflammatory cytokines; mitophagy inhibition abolishes protection, while STING inhibition restores it.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 3yesnono10.1111/acel.70701
11Grok 4.5R42805954H2What causal mechanism contributes to the age-related decline of thermogenic adipocytes in subcutaneous white adipose tissue?Age-related loss of sympathetic innervation density and norepinephrine release in subcutaneous white adipose tissue causally impairs β3-adrenergic recruitment of thermogenic (beige) adipocytes. Restoring local catecholaminergic tone would therefore rescue cold- or agonist-induced browning in aged animals. Mechanism: Progressive sympathetic axon rarefaction and reduced vesicular norepinephrine availability lower β3-AR–cAMP–PKA signaling in adipocyte progenitors and mature adipocytes, preventing UCP1 induction and beige differentiation. Prediction: Aged scWAT will show reduced tyrosine hydroxylase+ fiber density and lower tissue norepinephrine; local β3-agonist delivery or chemogenetic reactivation of adipose-projecting sympathetic neurons will restore UCP1+ beige adipocytes and cold-induced energy expenditure without requiring systemic endocrine rescue.Microglial ALKBH5 suppresses Ntf5 expression by demethylating Ntf5 mRNA, thereby limiting activation of PVH TrkB-positive neurons, sympathetic innervation of subcutaneous white adipose tissue, and adipocyte browning. Microglia-specific ALKBH5 knockout preserved brown adipocytes and improved metabolic activity during ageing in male mice.GPT-5.6 Sol 3; Claude Opus 5.5 3; Gemini 4 Argon 2yesyesno10.1038/s41467-026-77050-w
12Grok 4.5R42810392H3Can direct pharmacological activation of a conserved metabolic target extend lifespan across evolutionarily diverse organisms and induce longevity-associated changes in mammals?Selective pharmacological activation of AMPK is sufficient to extend lifespan in evolutionarily distant invertebrates and to induce longevity-associated physiological and molecular signatures in mammals. The longevity benefit is caused by AMPK itself rather than off-target pharmacology of multi-target drugs. Mechanism: Direct AMPK activation increases catabolic programs (fatty-acid oxidation, autophagy, mitochondrial biogenesis) and dampens anabolic growth signaling, shifting cells into a conserved energy-sparing, stress-resistant state that slows aging. Prediction: A highly selective AMPK agonist will extend lifespan in C. elegans and Drosophila and, in mice, raise AMPK activity, improve metabolic healthspan markers, and induce transcriptomic/proteomic aging-delay signatures, whereas chemically related inactive analogs will not.Direct pharmacological activation of AMPK with compound 991 extends lifespan in Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe. In mice, 991 induces a proteomic profile associated with pro-longevity biology, supporting the translational potential of AMPK activation.GPT-5.6 Sol 4; Claude Opus 5.5 5; Gemini 4 Argon 4yesnoyes10.1111/acel.70721
13Grok 4.5R42842666H1How do the different cellular compartments of the vascular wall causally contribute to distinct features of Hutchinson-Gilford progeria vascular pathology?Progerin expression specifically in vascular smooth muscle cells is sufficient to cause medial VSMC depletion and collagen-rich extracellular-matrix accumulation, independently of endothelial or adventitial progerin. Mechanism: Cell-autonomous nuclear lamina disruption in VSMCs triggers DNA damage responses, senescence and apoptosis, while dysregulating matrix gene programs that favor collagen deposition and medial remodeling. Prediction: Mice or vessel organoids with VSMC-restricted LMNA G609G (or equivalent) will reproduce medial VSMC loss and collagen accumulation, whereas endothelium- or adventitia-restricted progerin will not.In trilayer engineered blood vessels, HGPS fibroblasts causally promoted extracellular-matrix dysregulation and fibrotic signaling, while HGPS smooth muscle cells drove collagen accumulation and endothelial cells drove inflammation. Correcting the HGPS-associated mutation by base editing restored many pathological characteristics toward healthy levels.GPT-5.6 Sol 3; Claude Opus 5.5 3; Gemini 4 Argon 3yesnono10.1126/sciadv.aeh3785
14Grok 4.5R42844664H3Through what causal mechanism does time-restricted feeding protect against accelerated cardiovascular ageing?Time-restricted feeding protects against accelerated cardiovascular ageing by restoring intestinal epithelial barrier integrity, thereby limiting chronic low-grade endotoxemia that drives endothelial dysfunction and arterial stiffening. The barrier restoration is necessary and sufficient for the vascular benefit under otherwise matched caloric intake. Mechanism: Extended daily fasting upregulates tight-junction proteins (e.g., occludin, claudin-1) and mucus production via circadian and AMPK-dependent pathways in enterocytes; reduced translocation of LPS and other microbial products lowers TLR4/NF-κB signaling in endothelium and vascular smooth muscle, slowing hypertension, stiffening, and maladaptive remodeling. Prediction: In aged or progeroid models, TRF that improves gut barrier markers and lowers plasma LPS will prevent arterial stiffening and endothelial dysfunction, whereas TRF combined with experimental barrier disruption (or chronic low-dose LPS infusion matched to ad libitum levels) will abolish protection despite preserved feeding-window compliance and body weight.Time-restricted feeding enriches acetate-producing Limosilactobacillus, and microbiota-derived acetate is necessary and sufficient for its protection against angiotensin II-induced cardiovascular ageing. Acetate activates GPR43, inhibits HDAC3, increases H4K8 acetylation, and suppresses NLRP3-mediated pyroptosis and cellular senescence.GPT-5.6 Sol 2; Claude Opus 5.5 1; Gemini 4 Argon 2nonono10.1111/acel.70728
15Grok 4.5R42858183H1Can a defined intervention preserve lysosomal function and protein homeostasis during aging, thereby extending lifespan and maintaining organismal function, and through what mechanism?Chronic low-dose Tor inhibition preserves lysosomal acidification and autophagic flux during aging, which sustains protein homeostasis and extends lifespan. The benefit depends on maintained lysosomal protease activity rather than reduced protein synthesis alone. Mechanism: Reduced mTORC1 signaling derepresses TFEB/HLH-30 nuclear translocation, upregulating lysosomal biogenesis and v-ATPase subunits, thereby keeping lumenal pH low and clearance competent with age. Prediction: Genetic or pharmacological blockade of TFEB/HLH-30 or v-ATPase will abolish lifespan extension and lysosomal pH preservation under Tor inhibition, while matching the translation-rate reduction without Tor inhibition will not restore lysosomal competence.20(S)-ginsenoside Rg3 extended lifespan and preserved physiological and neuronal function in Caenorhabditis elegans by restoring lysosomal activity and autophagic flux. Rg3 remodeled neuronal lysosomal phosphoinositide signatures toward increased PI(4)P and reduced PI(3)P, suppressed RSKS-1/S6K–TORC1 signaling, and promoted nuclear HLH-30/TFEB activity, thereby improving proteostasis and resistance to polyglutamine- and α-synuclein-associated toxicity.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 3yesnono10.1111/acel.70755
16Grok 4.5R42669067H1How does SIRT1 regulate L1 retrotransposon activity, and does this regulation affect cellular senescence?SIRT1 maintains L1 silencing by deacetylating histone H3K9/H4K16 at L1 promoter and 5' UTR chromatin; decline of SIRT1 activity during senescence causes L1 transcriptional derepression that accelerates the senescence program. Mechanism: NAD+-dependent local chromatin deacetylation by SIRT1 preserves repressive chromatin at L1 loci; loss of this mark permits RNA polymerase II access and L1 mRNA production. Prediction: ChIP-seq will show increased H3K9ac/H4K16ac and reduced H3K9me3 specifically at L1 5' UTRs after SIRT1 knockdown or catalytic inhibition, preceding elevated L1 RNA and earlier SA-β-gal/p16 induction; a L1-promoter-targeted deacetylase tether will rescue silencing without restoring global SIRT1 activity.SIRT1 suppresses L1 transcription and retrotransposition by promoting the association and recruitment of Lamin B1 and KAP1 at the L1 5′-UTR, increasing repressive H3K9me3 chromatin. SIRT1 deficiency derepresses L1, activates cGAS–STING signaling, and induces cellular senescence; reverse-transcriptase inhibition with 3TC rescues this phenotype.GPT-5.6 Sol 3; Claude Opus 5.5 3; Gemini 4 Argon 3yesnono10.1111/acel.70689
17Grok 4.5R42811065H1Which local mechanism connects stress-induced sympathetic activity to cellular senescence and impaired osteogenesis, and can it be modulated non-invasively?Chronic psychological stress elevates bone-marrow norepinephrine from sympathetic terminals, which acts through β2-adrenergic receptors on mesenchymal stem/progenitor cells to drive p53/p21-dependent cellular senescence and thereby suppress osteogenic differentiation. Non-invasive peripheral neuromodulation that reduces local norepinephrine tone or blocks β2 signaling will reverse marrow senescence and restore bone formation. Mechanism: Sympathetic norepinephrine → β2-AR → cAMP/PKA → ROS/p53/p21 senescence program in osteoprogenitors Prediction: Marrow β2-AR knockout or selective β2 blockade will abolish stress-induced senescence markers (p16, SA-β-gal) and rescue osteogenesis even when circulating catecholamines remain high, whereas β1-selective blockade will not; peripheral nerve stimulation that lowers marrow NE will phenocopy β2 blockade.Sympathetic neuropeptide Y drives stress-induced osteoporosis by activating adipocyte Y1R and inducing senescence in bone marrow adipocytes, which impairs osteogenesis. Pulsed electromagnetic fields activate sensory nerves, causing local semaphorin 3A release that suppresses sympathetic neuropeptide Y secretion through a peripheral circuit independent of central pathways.GPT-5.6 Sol 2; Claude Opus 5.5 2; Gemini 4 Argon 2nonono10.1038/s41467-026-77654-2
18Grok 4.5R42806003H1What intercellular mechanism causes placental senescence during maternal ageing, and can disrupting this mechanism restore placental and offspring health?Maternal ageing elevates CD38 on decidual and circulating immune cells, depleting NAD+ in the shared placental microenvironment and thereby suppressing SIRT1/PARP activity in trophoblasts, which accelerates placental cellular senescence and metabolic failure. Restoring NAD+ or inhibiting maternal CD38 should reverse placental senescence and improve fetal growth. Mechanism: Maternal CD38-driven NAD+ depletion creates a metabolic niche that forces trophoblast senescence. Prediction: Placental NAD+ and SIRT1 activity fall with maternal age in parallel with CD38+ immune infiltration; CD38 inhibition or NAD+ precursors restore trophoblast NAD+/SIRT1, reduce senescence markers, and improve fetal outcomes even when maternal chronological age is unchanged.Early inflammation and senescence-associated secretory phenotype activity induce IRF5-dependent CD38 expression in macrophages, which depletes NAD+ in decidual stromal cells and thereby promotes age-related placental senescence. Genetic or pharmacological disruption of this axis, including CD38 inhibition or NAD+ precursor treatment, reduces placental senescence, rescues intrauterine growth restriction, and improves offspring metabolic outcomes.GPT-5.6 Sol 3; Claude Opus 5.5 4; Gemini 4 Argon 4yesnoyes10.1038/s41467-026-77217-5
19Grok 4.5R42806572H2Can a dose-titratable immune engager safely eliminate senescent cells and alleviate age-related pathology across mice and non-human primates, and what measurement can guide its safe dosing?A dose-titratable bispecific immune engager that redirects T cells to a senescence-associated surface antigen will clear a therapeutically relevant fraction of senescent cells in aged mice and non-human primates at exposures below those that trigger systemic cytokine release. Circulating IL-6 and IFN-γ rise steeply only after the efficacious dose window, so their early kinetics can guide safe titration. Mechanism: Target engagement on senescent cells forms an immunological synapse that drives selective cytotoxicity; off-target or excess engagement expands systemic cytokine production once a threshold of T-cell activation is crossed. Prediction: In both species, intermediate engager doses will reduce senescence markers (p16INK4a, SA-β-gal, SASP factors) and improve tissue function without grade ≥2 cytokine release, while cytokine AUC will increase nonlinearly only at higher doses that do not further improve efficacy.A GFD-CD3 bispecific T-cell engager targeting uPAR-positive senescent cells caused dose-dependent senolysis and alleviated age-related pathologies in aged mice and non-human primates at low doses. High doses caused hepatotoxicity through T-cell attack on hepatic endothelial cells, while serum aminotransferase monitoring enabled dosing that retained senolytic efficacy without observed adverse effects.GPT-5.6 Sol 3; Claude Opus 5.5 3; Gemini 4 Argon 3yesnono10.1111/acel.70742
20Gemini 4 ArgonR42605193H3Which circulating regulator causally promotes cellular senescence and organismal ageing, and through what intracellular signaling mechanism does it act?Age-related elevation of circulating interleukin-11 (IL-11) causally promotes multi-organ cellular senescence, metabolic dysfunction, and organismal ageing. Secreted progressively by aged hepatocytes, adipocytes, and stromal cells, circulating IL-11 coordinates systemic tissue decline by simultaneously driving pro-senescent anabolic signaling and suppressing cellular energy-sensing pathways. Mechanism: Circulating IL-11 binds the cell-surface IL-11Rα/gp130 receptor complex in target parenchymal and stromal cells to activate MEK/ERK1/2, which phosphorylates and inactivates LKB1 (at Ser428) to suppress AMPK while concurrently activating mTORC1–S6K1 and canonical JAK2/STAT3 signaling. This dual ERK–mTORC1 activation and AMPK inhibition halts autophagic and mitophagic flux, increases mitochondrial reactive oxygen species (ROS), and transcriptionally upregulates p16INK4a, p21CIP1, and NF-κB-dependent senescence-associated secretory phenotype (SASP) factors. Prediction: Systemic administration of neutralizing anti-IL-11 or anti-IL-11Rα antibodies, or adult-onset Il11ra1 deletion, in middle-aged mice will restore tissue AMPK activity, suppress mTORC1 and STAT3 hyperactivation, reduce multi-organ p16INK4a/p21CIP1 senescence burden, reverse age-related insulin resistance and hepatic steatosis, and extend median and maximum lifespan; conversely, chronic IL-11 infusion in young mice will induce widespread senescence that is blocked by pharmacological ERK1/2 or mTORC1 inhibition.ANGPTL8 directly interacts with AKT2 and activates the AKT–mTOR–S6K pathway, thereby driving cell-autonomous adipocyte senescence. Deleting Angptl8 in mice reduces adipose senescence, delays age-related functional decline, and extends lifespan, while genetic or pharmacological pathway inhibition abolishes ANGPTL8-induced senescence.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 2nonono10.1111/acel.70671
21Gemini 4 ArgonR42608398H3Can a microbiota-derived factor directly cause the cellular dysfunction and progressive tissue remodeling characteristic of idiopathic pulmonary fibrosis, and if so, through what mechanism?A conserved pro-apoptotic peptide proteolytically cleaved from bacterial cell-wall transglycosylases (such as corisin derived from lung-resident Staphylococcus and related commensal bacteria) directly initiates progressive pulmonary fibrosis without requiring live infection. This microbiota-derived peptide acts on alveolar epithelial cells to simultaneously trigger apoptosis, cellular senescence, and epithelial-to-mesenchymal phenotypic transitions. Mechanism: Extracellular bacterial serine proteases cleave conserved regions of bacterial transglycosylases to release a bioactive peptide into the alveolar space. Upon internalization or receptor engagement on alveolar type II (AT2) epithelial cells, the peptide disrupts mitochondrial membrane potential and increases mitochondrial reactive oxygen species, triggering intrinsic caspase-3-mediated apoptosis in a subset of cells while activating ATM/p53/p21 DNA damage signaling, senescence-associated secretory phenotype (SASP) release, and TGF-beta1/SMAD- and Snail-mediated epithelial-to-mesenchymal transition (EMT) in surviving cells to drive myofibroblast accumulation and extracellular matrix deposition. Prediction: Intratracheal instillation of the purified synthetic bacterial peptide alone into uninjured mice will be sufficient to induce AT2 apoptosis, p21/SA-beta-gal-positive senescence, EMT marker expression, and progressive collagen deposition, whereas protease-resistant removal of peptidic factors or treatment with a peptide-neutralizing monoclonal antibody will completely abolish these cell-fate abnormalities and fibrotic remodeling.The microbiota-derived peptide corisin directly disrupts epithelial proteostasis by targeting and enhancing the ubiquitin-proteasome system, thereby inducing apoptosis, cellular senescence, epithelial-mesenchymal transition, and fibrotic remodeling. Depleting native corisin from patient bronchoalveolar lavage fluid removes its proapoptotic activity, while constitutive corisin expression in mice causes spontaneous progressive pulmonary fibrosis and worsens bleomycin-induced injury and mortality.GPT-5.6 Sol 3; Claude Opus 5.5 4; Grok 4.7 3yesnono10.1038/s41467-026-76162-7
22Gemini 4 ArgonR42627902H5Which regulatory mechanism drives the age-associated shift away from erythroid differentiation and toward platelet-producing lineage programs?Age-associated upregulation and chromatin pioneering of the transcription factor FLI1 in hematopoietic stem and megakaryocyte-erythroid progenitor cells (MEPs) directly activates platelet-lineage gene programs while competitively repressing KLF1-driven erythroid differentiation. This shift in the FLI1-to-KLF1 transcriptional balance causally redirects bipotent progenitors and stem cells toward megakaryocytopoiesis during ageing. Mechanism: During ageing, increased accessibility at ETS-family binding motifs allows FLI1 (in cooperation with RUNX1 and GATA2) to occupy and activate megakaryocytic enhancers (such as MPL, ITGA2B, and GP1BA) while physically sequestering GATA1 away from KLF1 and recruiting corepressors to erythroid-specific loci (such as EPOR and HBB), thereby blocking erythroid commitment and promoting direct megakaryocyte generation. Prediction: Single-cell ATAC/RNA-seq of aged HSCs and MEPs will show selective enrichment of open FLI1/ETS motifs prior to lineage commitment, and partial genetic knockdown or degron-mediated reduction of FLI1 in aged progenitors will restore KLF1 target gene expression and normalize the erythroid-to-platelet output ratio even in the presence of an aged inflammatory bone marrow niche.Pbx1 is a regulatory hub that drives age-related transcriptional and differentiation changes in hematopoietic stem cells. It suppresses erythroid differentiation by repressing Gata1 expression, thereby contributing to the lineage imbalance characteristic of HSC ageing.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 2nonono10.1126/sciadv.aeb1346
23Gemini 4 ArgonR42647119H4How does aging affect injury-induced neuronal reprogramming in the retina, and what mechanisms account for any age-related loss of regenerative capacity?Aging impairs injury-induced retinal neuronal reprogramming by priming resident microglia toward a persistent pro-inflammatory state that locks Müller glia into reactive gliosis rather than neurogenesis. Chronic inflammatory signaling after injury in aged retinas actively antagonizes proneural transcription factor activity in Müller glia. Mechanism: In aged retinas, senescent and dystrophic microglia respond to injury with sustained secretion of TNF-alpha, IL-6, and IL-1beta, driving prolonged hyperactivation of JAK/STAT3 and NF-kappaB signaling within Müller glia. Phosphorylated STAT3 and RelA/p65 directly compete with proneural pioneer factors such as Ascl1 for transcriptional co-activators and promote expression of gliogenic and inhibitory factors (such as Id1/3, Socs3, and Gfap), blocking the transition from reactive glia to neurogenic progenitors. Prediction: Microglial depletion (e.g., via CSF1R inhibition) or Müller glia-specific inhibition of STAT3/NF-kappaB signaling in aged injured retinas will restore Ascl1-driven neurogenic reprogramming efficiency to youthful levels, even without directly altering the intrinsic epigenetic landscape of Müller glia prior to injury.Aging suppresses Müller glia-to-neuron reprogramming by preventing activation of progenitor programs while promoting reactive and inflammatory glial states and an exaggerated retinal neuroimmune response. Dexamethasone partially restores neurogenesis, confirming that age-associated inflammation is a causal, therapeutically tractable component of the regenerative defect, alongside glial-intrinsic barriers.GPT-5.6 Sol 4; Claude Opus 5.5 4; Grok 4.7 4yesnoyes10.1073/pnas.2612369123
24Gemini 4 ArgonR42647635H4Does experimentally increasing somatic mitochondrial DNA mutation burden cause premature ageing and shortened lifespan in a large mammal?Experimentally increasing the somatic mtDNA mutation rate via proofreading-deficient DNA polymerase gamma (POLG) in a large mammal directly drives systemic premature ageing and shortens lifespan by exhausting adult stem and progenitor cell pools. This progeroid phenotype arises even without elevated mitochondrial reactive oxygen species (ROS). Mechanism: Accumulation of random point mutations and linear deletions in mtDNA destabilizes respiratory chain complexes I and IV, triggering bioenergetic failure and intrinsic caspase-dependent apoptosis specifically within rapidly dividing somatic stem cell compartments (such as hematopoietic, intestinal, and neural progenitors), thereby impairing tissue regeneration. Prediction: Large mammals engineered with exonuclease-deficient POLG will exhibit early-onset alopecia, kyphosis, anemia, sarcopenia, and reduced lifespan accompanied by severe depletion of tissue-resident stem cells and respiratory chain complex instability, but without a corresponding increase in oxidative macromolecular damage (e.g., 8-oxodG or lipid peroxidation) or rescue by mitochondrial antioxidants.Prime-edited pigs expressing proofreading-deficient POLG accumulated elevated somatic mitochondrial DNA mutations and developed systemic premature-ageing phenotypes. These included weight loss, rough hair, anaemia, tissue abnormalities, increased apoptosis, elevated senescence-associated markers, and shortened lifespan.GPT-5.6 Sol 4; Claude Opus 5.5 5; Grok 4.7 4yesnoyes10.1126/sciadv.adu0632
25Gemini 4 ArgonR42663085H1Can an intrinsic molecular regulator be enhanced to reactivate quiescent hair follicles and restore hair regeneration, and through what mechanism?Genetic or pharmacological induction of intrinsic microRNA-205 (miR-205) expression in aged hair follicle stem cells (HFSCs) reactivates quiescent telogen follicles and restores anagen hair regeneration. This reactivation occurs by reversing age-associated cellular stiffening in the bulge and hair germ compartments. Mechanism: miR-205 directly binds and post-transcriptionally represses transcripts encoding RhoA-ROCK pathway activators and actomyosin regulators (including Arhgef11 and Plekhg5), reducing myosin light chain II phosphorylation (p-MLC2) and cortical F-actin polymerization, which lowers intracellular mechanical tension and permits cell-cycle re-entry. Prediction: Atomic force microscopy will show reduced cortical stiffness in aged HFSCs upon miR-205 induction prior to proliferation, and hair regeneration will be phenocopied by ROCK/myosin II inhibition (e.g., blebbistatin) or abolished by expressing miR-205-resistant Arhgef11 without altering upstream BMP or Wnt ligand levels.Increasing TPP1, either through stem-cell Acd overexpression or topical stabilization with the peptide TELODIN, accelerates hair regeneration in mice. TPP1 coordinates telomere capping and telomerase recruitment, activating bulge hair follicle stem cells and promoting their proliferation, differentiation, and migration as follicles transition synchronously from telogen to anagen.GPT-5.6 Sol 2; Claude Opus 5.5 0; Grok 4.7 2nonono10.1111/acel.70692
26Gemini 4 ArgonR42664348H5By what causal molecular mechanism does excess adiposity promote osteoarthritis progression, and can disrupting that mechanism reduce joint pathology?Excess adipose tissue promotes osteoarthritis progression by secreting complement factor D (adipsin) into the circulation, which acts as the rate-limiting protease to activate the alternative complement cascade within synovial fluid and drive complement-mediated joint injury. Genetic deletion or pharmacological inhibition of circulating adipsin will protect against obesity-induced cartilage degeneration and synovitis. Mechanism: Adipocytes serve as the primary endocrine source of circulating adipsin (CFD), which diffuses into the joint space and cleaves factor B complexed with C3b to form the alternative pathway C3 convertase (C3bBb); subsequent cascade amplification generates C3a/C5a anaphylatoxins and deposits sublytic membrane attack complexes (C5b-9) on chondrocytes and synovial macrophages, activating C5aR1 and MAPK/ERK signaling to induce catabolic metalloproteinases and synovial inflammation. Prediction: Cfd-deficient mice or lipodystrophic mice fed an obesogenic diet will be protected from synovial C3b/C5b-9 deposition and osteoarthritis, and surgical transplantation of wild-type—but not Cfd-knockout—adipose tissue into fat-free mice will restore synovial alternative complement activation and susceptibility to joint degeneration.Adipocyte-derived extracellular vesicles enriched in mitochondrial components transfer mitochondrial DNA to chondrocytes, where it activates cGAS-STING signaling and causes inflammation, metabolic dysfunction, senescence, and cartilage degeneration. Genetic or pharmacological inhibition of this pathway attenuates cartilage damage, pain behaviors, and gait abnormalities.GPT-5.6 Sol 2; Claude Opus 5.5 0; Grok 4.7 2nonono10.1126/sciadv.aee6780
27Gemini 4 ArgonR42665751H4How does age-associated mitochondrial dysfunction initiate and propagate inflammatory signaling that causes ovarian functional decline?Age-associated loss of mitochondrial membrane integrity in granulosa cells and oocytes causes cytosolic leakage of mitochondrial DNA (mtDNA), activating the cGAS-STING pathway to initiate sterile follicular inflammation. Intercellular transfer of 2'3'-cGAMP through connexin gap junctions and paracrine type I interferon/NF-kB cytokine secretion propagate this inflammatory state across the cumulus-oocyte complex and ovarian stroma, driving follicular atresia and endocrine decline. Mechanism: Age-related decline in TFAM stoichiometry and cristae remodeling promote VDAC1 oligomerization and BAX/BAK macropore formation on the outer mitochondrial membrane, releasing mtDNA fragments into the cytosol. Cytosolic mtDNA binds cGAS to synthesize 2'3'-cGAMP, which activates STING-TBK1-IRF3/NF-kB signaling within the stressed cell and diffuses through Connexin-43 and Connexin-37 gap junctions at transzonal projections into adjacent follicular cells, amplifying IL-6, TNF-alpha, CXCL10, and IFN-beta production throughout the follicle. Prediction: Oocyte- or granulosa-specific genetic deletion or pharmacological inhibition of STING (e.g., H-151) or gap-junctional cGAMP transfer in aged mice will abolish pan-follicular interferon-stimulated gene expression and preserve follicular reserve and estradiol production, even when mitochondrial bioenergetic dysfunction or NLRP3 inflammasome competence remains intact.Ageing-associated mitochondrial DNA leakage from oocytes activates oocyte cGAS, generating cGAMP that triggers STING signaling and passes through gap junctions to activate STING in surrounding granulosa cells. Genetic deletion of oocyte Cgas or pharmacological STING inhibition ameliorates inflammation and ovarian dysfunction, establishing this signaling axis as a causal driver of ovarian ageing.GPT-5.6 Sol 4; Claude Opus 5.5 5; Grok 4.7 5yesnoyes10.1038/s43587-026-01195-y
28Gemini 4 ArgonR42689491H5Can in vivo correction of the HGPS-causing mutation rescue molecular, cellular, and structural defects in bone, and how does treatment timing affect efficacy?In vivo correction of the HGPS-causing LMNA mutation rescues molecular and cellular osteoblast defects regardless of age, but complete restoration of bone microarchitecture and biomechanical strength requires early intervention before osteoprogenitor exhaustion and growth plate senescence occur. Late treatment achieves only partial structural rescue because recovery is rate-limited by slow adult bone remodeling turnover and a depleted mesenchymal progenitor pool. Mechanism: Eliminating progerin production and restoring wild-type lamin A/C repairs nuclear lamina architecture and relieves p53/p21-mediated cell-cycle arrest in surviving osteoprogenitors and osteoblasts. When correction occurs during early postnatal growth, abundant mesenchymal stem and progenitor cells rapidly populate modeling bone surfaces to normalize cortical thickness and trabecular volume; when initiated after skeletal maturity, irreversible senescence of chondro-osseous progenitors and low baseline remodeling rates restrict the replacement of pre-existing dysplastic bone matrix. Prediction: Both early and late genetic correction will normalize nuclear morphology and osteogenic gene expression in active osteoblasts within weeks, whereas micro-CT cortical bone volume and three-point bending fracture resistance will fully normalize only in the early-treated cohort, with late-treated animals showing a rescue trajectory proportional to bone formation rate and residual Sca-1+/PDGFRa+ progenitor frequency.Adenine base editing of the pathogenic LMNA variant reduced progerin expression, restored extracellular-matrix deposition and mineralization in progeroid osteoblasts, and partially rescued bone structure and physical properties in mice. In vivo correction was strongly age-dependent, with early postnatal delivery producing substantially greater editing and skeletal benefit than later treatment.GPT-5.6 Sol 4; Claude Opus 5.5 4; Grok 4.7 3yesnoyes10.1111/acel.70700
29Gemini 4 ArgonR42723546H4Can a cell-free regenerative treatment alleviate UV-induced skin photoaging, and which causal intracellular mechanism mediates its protective effects?Cell-free regenerative therapy (such as stem cell-derived extracellular vesicles) alleviates UV-induced skin photoaging by activating PINK1/Parkin-mediated mitophagy to clear damaged mitochondria. This targeted organelle clearance prevents the leakage of mitochondrial DNA (mtDNA) into the cytosol, thereby suppressing activation of the cGAS–STING innate immune pathway and downstream senescence-associated secretory phenotype (SASP) inflammation. Mechanism: Bioactive cargo within the cell-free treatment stimulates PINK1 stabilization and Parkin recruitment to depolarized mitochondria in UV-irradiated keratinocytes and dermal fibroblasts, promoting their autophagic degradation. By eliminating dysfunctional mitochondria before outer membrane permeabilization or mPTP opening can occur, the treatment prevents cytosolic accumulation of oxidized mtDNA, blocking cGAS recognition, 2'3'-cGAMP synthesis, STING/TBK1/IRF3/NF-κB phosphorylation, and the resulting sterile inflammatory and senescent state. Prediction: Genetic depletion of PINK1/Parkin or pharmacological inhibition of mitophagy in UV-exposed skin models will abolish the therapy's ability to reduce cytosolic mtDNA levels, STING phosphorylation, SASP cytokine secretion, and p16/p21 expression, whereas exogenous STING agonists (e.g., DMXAA or cGAMP) will override the protective effects despite intact mitochondrial clearance.Human umbilical cord mesenchymal stem cell secretome protects against UV-induced photoaging by enhancing mitophagy, which clears damaged mitochondria and reduces cytosolic mitochondrial DNA leakage. This suppresses cGAS-STING signaling and downstream inflammatory cytokines; mitophagy inhibition abolishes protection, while STING inhibition restores it.GPT-5.6 Sol 4; Claude Opus 5.5 5; Grok 4.7 4yesnoyes10.1111/acel.70701
30Gemini 4 ArgonR42736449H4Can an unannotated protein product that is altered in Alzheimer's disease causally regulate cellular function relevant to disease pathology?Disease-associated dysregulation of an unannotated small open reading frame (sORF)-encoded microprotein localized to the inner mitochondrial membrane causally impairs oxidative phosphorylation and drives bioenergetic failure in Alzheimer's disease neurons independently of the canonical protein encoded at the same locus. Mechanism: Uncoupled translational regulation or transcript isoform switching in Alzheimer's disease alters the abundance of a mitochondrial-targeted microprotein encoded by an upstream or overlapping reading frame. This microprotein normally acts as an assembly or stabilizing factor for electron transport chain supercomplexes, and its perturbation in disease disrupts Complex I/IV activity, reduces ATP synthesis, and elevates mitochondrial reactive oxygen species (mtROS). Prediction: Selective genetic restoration or ablation of the unannotated microprotein in neuronal models—while holding canonical host protein expression constant—directly modifies basal and maximal oxygen consumption rates, mitochondrial membrane potential, and mtROS levels, and affinity purification-mass spectrometry confirms its physical interaction with respiratory chain subunits.A small open reading frame at the MKKS locus encodes a 63-amino-acid microprotein that is the locus's predominant translation product and is downregulated in Alzheimer's disease. Experimental loss of this microprotein impairs mitochondrial respiration in microglia, supporting a causal role in microglial bioenergetics.GPT-5.6 Sol 3; Claude Opus 5.5 3; Grok 4.7 3yesnono10.1038/s43587-026-01207-x
31Gemini 4 ArgonR42775695H4What causes defective mitochondrial quality-control flux in the aging heart, and how can restoring mitochondrial homeostasis reverse cardiac aging?Age-associated hyperactivation of cardiac mTORC1 and upregulation of the Beclin-1 inhibitor Rubicon, combined with impaired TFEB nuclear translocation, block late-stage mitophagosome-lysosome fusion and lysosomal acidification in cardiomyocytes, stalling mitophagy flux mid-stream. Therapeutically restoring lysosomal biogenesis and autolysosomal fusion clears accumulated mitophagosomal intermediates, suppresses cytosolic mtDNA sensing, and reverses age-related diastolic and systolic dysfunction. Mechanism: In aged cardiomyocytes, chronic nutrient and stress signaling elevates mTORC1 activity and Rubicon expression, which inhibits the UVRAG-Vps34 complex and retains TFEB in the cytosol while diminishing v-ATPase-dependent lysosomal acidification. Because early mitochondrial damage continues to trigger priming and autophagosomal engulfment against a downstream bottleneck in Rab7/SNARE-mediated lysosomal fusion and proteolysis, un-degraded mitophagosomes accumulate, leak oxidized mtDNA into the cytosol, and activate cGAS-STING and NLRP3 inflammatory signaling. Prediction: Dual-fluorescent mitophagy reporter mice (such as mt-Keima or mito-QC) will show an increased ratio of neutral-pH mitophagosomes to acidic mitolysosomes in aged hearts rather than a lack of autophagosomal sequestration; furthermore, stimulating early mitophagy initiation alone will exacerbate cardiomyocyte toxicity, whereas Rubicon depletion or TFEB activation will restore acidic mitolysosome formation and rescue ventricular function.Age-associated upregulation of HIF-3α drives excessive BNIP3 expression in senescent cardiomyocytes, causing mitophagy flux congestion and cardiac aging. Mitochondrial transplantation restores intracellular ATP homeostasis, restrains pathological HIF-3α–BNIP3 signaling, normalizes mitophagy flux, and rejuvenates the aging heart.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 2nonono10.1111/acel.70720
32Gemini 4 ArgonR42805954H5What causal mechanism contributes to the age-related decline of thermogenic adipocytes in subcutaneous white adipose tissue?Age-related decline in subcutaneous white adipose tissue (scWAT) thermogenesis is driven by the loss of adipocyte- and stromal-derived neurotrophic factors (such as NGF, BDNF, and S100B) alongside increased repulsive axon guidance cues (such as SEMA3A), causing structural retraction of sympathetic nerve arborizations. Without dense peri-adipocyte sympathetic innervation, environmental cold stimuli fail to deliver sufficient local norepinephrine to recruit beige adipocytes. Mechanism: Transcriptional and epigenetic remodelling of ageing adipocytes and PDGFRalpha+ stromal progenitors downregulates retrograde neurotrophic signals (NGF/TrkA and CLSTN3B-S100B axes) while upregulating semaphorin-neuropilin repulsive signalling, leading to axonal die-back of tyrosine hydroxylase-positive (TH+) sympathetic fibres and physical uncoupling of the neuro-adipose junction. Prediction: 3D whole-mount immunolabelling of aged scWAT will reveal a marked reduction in TH+ sympathetic nerve fibre density and branching prior to losing intrinsic adipocyte responsiveness to exogenous beta-3-adrenergic agonists (CL316,243), and viral restoration of NGF or S100B in aged scWAT will regenerate axonal arbors and restore cold-induced UCP1+ beige adipocyte formation.Microglial ALKBH5 suppresses Ntf5 expression by demethylating Ntf5 mRNA, thereby limiting activation of PVH TrkB-positive neurons, sympathetic innervation of subcutaneous white adipose tissue, and adipocyte browning. Microglia-specific ALKBH5 knockout preserved brown adipocytes and improved metabolic activity during ageing in male mice.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 2nonono10.1038/s41467-026-77050-w
33Gemini 4 ArgonR42810392H4Can direct pharmacological activation of a conserved metabolic target extend lifespan across evolutionarily diverse organisms and induce longevity-associated changes in mammals?Direct allosteric activation of the conserved energy sensor AMP-activated protein kinase (AMPK) is sufficient to extend lifespan across evolutionarily diverse invertebrates and induce caloric-restriction-like metabolic rejuvenation in mammals without requiring mitochondrial electron transport chain inhibition. Engaging AMPK directly via its allosteric binding pocket bypasses the pleiotropic off-target effects of indirect metabolic poisons to drive conserved pro-longevity programs. Mechanism: Selective small-molecule binding to the AMPK heterotrimeric complex (such as at the allosteric drug and metabolite [ADaM] site between the alpha-kinase domain and beta-carbohydrate-binding module) stabilizes the active enzyme conformation and prevents dephosphorylation of Thr172. Activated AMPK directly phosphorylates conserved downstream substrates—including ULK1 to initiate autophagy, ACC1/2 to promote fatty acid beta-oxidation, Raptor/TSC2 to restrain mTORC1 signaling, and FOXO/PGC-1alpha to enhance mitochondrial quality control and oxidative stress resistance. Prediction: Administration of a selective direct AMPK activator extends median and maximum lifespan in wild-type C. elegans and Drosophila and improves insulin sensitivity, autophagic flux, and transcriptomic age biomarkers in aged mice without altering cellular ATP/AMP ratios or complex I respiration, whereas genetic ablation of the catalytic AMPK alpha-subunit (aak-2 in worms, AMPKalpha in flies and mice) or point mutation of the allosteric drug-binding pocket completely abolishes both lifespan extension and mammalian metabolic benefits.Direct pharmacological activation of AMPK with compound 991 extends lifespan in Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe. In mice, 991 induces a proteomic profile associated with pro-longevity biology, supporting the translational potential of AMPK activation.GPT-5.6 Sol 3; Claude Opus 5.5 5; Grok 4.7 4yesnoyes10.1111/acel.70721
34Gemini 4 ArgonR42826184H1What causal molecular mechanism remodels three-dimensional genome organization during ageing and thereby promotes age-associated inflammatory and activation-related transcription?Chronic age-associated stress and metabolic signaling induce persistent chromatin engagement of pioneer and inducible transcription factors—principally AP-1 (FOS/JUN) and NF-κB—which actively redistribute SWI/SNF (BAF) chromatin remodelers and cohesin complexes to form de novo enhancer-promoter loops at inflammatory and activation-related loci. This factor-driven recruitment sequesters architectural loop-extrusion machinery away from homeostatic lineage-identity super-enhancers, simultaneously promoting basal activation programs and impairing specialized immune function. Mechanism: Persistent low-grade signaling stabilizes AP-1 complexes at latent distal enhancers, where they recruit BAF remodelers to open chromatin and anchor cohesin-Mediator complexes, establishing de novo long-range chromatin loops with cytokine and activation promoters at the expense of homeostatic cell-identity loops. Prediction: Acute genetic or pharmacological inhibition of AP-1 DNA binding or BAF ATPase activity in aged cells will selectively collapse age-acquired enhancer-promoter loops at inflammatory loci in H3K27ac/cohesin HiChIP assays, redistribute cohesin back to homeostatic identity loci, and reverse the basal activation transcriptional signature.Age-associated reduction of SATB1 in naive CD4+ T cells weakens TAD boundaries and expands the range of CTCF-mediated interactions without changing CTCF occupancy, producing enhancer-promoter rewiring and increased expression of proinflammatory and proactivation genes. Conditional SATB1 deletion recapitulates these ageing-associated architectural and transcriptional changes, establishing SATB1 deficiency as a causal contributor.GPT-5.6 Sol 2; Claude Opus 5.5 1; Grok 4.7 2nonono10.1126/sciadv.aeg1622
35Gemini 4 ArgonR42842666H3How do the different cellular compartments of the vascular wall causally contribute to distinct features of Hutchinson-Gilford progeria vascular pathology?Progerin expression in medial vascular smooth muscle cells (VSMCs) and intimal endothelial cells (ECs) drives distinct pathological features in a strictly cell-autonomous, compartment-specific manner: mutant VSMCs directly generate extracellular-matrix (ECM) abnormalities and collagen accumulation, whereas mutant ECs independently drive vascular inflammation. Neither compartment requires the LMNA mutation in the neighbouring layer to manifest its signature pathology. Mechanism: In the tunica media, progerin-induced nuclear lamina stiffening disrupts LINC-complex mechanotransduction under cyclic stretch, shifting VSMCs from a contractile to a synthetic, profibrotic phenotype with elevated collagen and matrix metalloproteinase production. Concurrently, in the tunica intima, progerin impairs endothelial adaptation to laminar shear stress, directly activating NF-kappaB transcriptional programs and surface adhesion molecules (ICAM-1, VCAM-1) without altering mural matrix synthesis. Prediction: In heterotypic three-dimensional blood-vessel models or lineage-specific mosaic mice, vessels composed of HGPS VSMCs and wild-type ECs will exhibit medial ECM disorganization and collagen accumulation without endothelial inflammatory activation, whereas vessels with HGPS ECs and wild-type VSMCs will display robust intimal inflammation and leukocyte adhesion without medial fibrosis.In trilayer engineered blood vessels, HGPS fibroblasts causally promoted extracellular-matrix dysregulation and fibrotic signaling, while HGPS smooth muscle cells drove collagen accumulation and endothelial cells drove inflammation. Correcting the HGPS-associated mutation by base editing restored many pathological characteristics toward healthy levels.GPT-5.6 Sol 3; Claude Opus 5.5 4; Grok 4.7 3yesnono10.1126/sciadv.aeh3785
36Gemini 4 ArgonR42853938H4What splicing-dependent mechanism enables resistance to CDK4/6 inhibition, and can disrupting it restore therapy-induced senescence?Sustained activity of the protein arginine methyltransferase PRMT5 enables resistance to CDK4/6 inhibition by maintaining spliceosomal fidelity required for productive splicing of MDM4 and the histone methyltransferase EZH2. Pharmacological inhibition of PRMT5 or antisense oligonucleotide-mediated skipping of MDM4 exon 6 disrupts this axis, restoring p53 activation, H3K27me3 loss at senescence loci, and durable therapy-induced senescence. Mechanism: In drug-sensitive cells, CDK4/6 inhibition suppresses E2F-dependent expression of PRMT5 cofactors (such as WDR77/MEP50), reducing symmetric dimethylation of spliceosomal Sm proteins (SmD1/SmD3). This hypomethylation impairs recognition of weak 5' splice sites, causing exon 6 skipping in MDM4 (generating unstable MDM4-S subject to nonsense-mediated decay) and intron retention in EZH2, which together unleash p53-p21 signaling and derepress H3K27me3-silenced senescence genes. Resistant cells maintain high PRMT5-WDR77 activity independently of CDK4/6, preserving full-length MDM4 to degrade p53 and full-length EZH2 to epigenetically silence CDKN2A and SASP enhancers; blocking PRMT5 or directly forcing MDM4 exon 6 skipping re-establishes both p53 transcriptional activity and chromatin derepression required for irreversible arrest. Prediction: In CDK4/6 inhibitor-resistant cells with wild-type TP53, RNA-seq and anti-sDMA immunoprecipitation will reveal preserved Sm protein symmetric dimethylation alongside full-length MDM4 (exon 6 inclusion) and EZH2 transcripts, and combining palbociclib with a PRMT5 inhibitor or an MDM4 exon 6 splice-switching oligonucleotide—unlike inhibiting downstream CDK2 alone—will selectively restore SA-beta-gal activity and p21/SASP induction without requiring exogenous DNA damage.Increased inclusion of exon 2 drives SUV39H2 overexpression and CDK4/6 inhibitor resistance. Suppressing SUV39H2 expression or methyltransferase activity removes H3K9me3 from enhancer regions, opens associated promoters, activates p21 and other senescence-associated genes, and restores therapy-induced senescence.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 2nonono10.1126/sciadv.aea2001
37Gemini 4 ArgonR42858183H3Can a defined intervention preserve lysosomal function and protein homeostasis during aging, thereby extending lifespan and maintaining organismal function, and through what mechanism?Dietary supplementation with the polyamine spermidine preserves lysosomal proteolytic activity and maintains protein homeostasis during aging, thereby extending lifespan and organismal function, by sustaining the eIF5A-dependent translation of the master lysosomal transcription factor TFEB (HLH-30). Mechanism: Spermidine serves as the sole aminobutyl donor for the enzymatic hypusination of eukaryotic translation initiation factor 5A (eIF5A) via deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A resolves ribosome stalling at polyproline motifs within TFEB/HLH-30 mRNA, preventing age-related translational decline of TFEB and maintaining transcription of the Coordinated Lysosomal Expression and Regulation (CLEAR) network, including V-ATPase subunits and cathepsin hydrolases. Prediction: Spermidine treatment in aged organisms will restore nuclear TFEB/HLH-30 levels, lysosomal cathepsin activity, and clearance of insoluble ubiquitinated protein aggregates to extend lifespan, whereas pharmacological inhibition of DHPS (via GC7) or mutation of the eIF5A hypusination site (K50A) will completely abolish these benefits even when mTORC1 is pharmacologically inhibited to force nuclear translocation of existing TFEB.20(S)-ginsenoside Rg3 extended lifespan and preserved physiological and neuronal function in Caenorhabditis elegans by restoring lysosomal activity and autophagic flux. Rg3 remodeled neuronal lysosomal phosphoinositide signatures toward increased PI(4)P and reduced PI(3)P, suppressed RSKS-1/S6K–TORC1 signaling, and promoted nuclear HLH-30/TFEB activity, thereby improving proteostasis and resistance to polyglutamine- and α-synuclein-associated toxicity.GPT-5.6 Sol 3; Claude Opus 5.5 2; Grok 4.7 2noyesno10.1111/acel.70755
38Gemini 4 ArgonR42585340H5Which molecular mechanism enables mild cellular stress to preserve protein homeostasis during ageing and extend organismal lifespan?Mild thermal or proteotoxic stress extends lifespan and prevents age-related protein aggregation by co-activating heat shock factor 1 (HSF-1) and the transcription factor TFEB/HLH-30 to transcriptionally upregulate the macroautophagy-lysosome machinery. Rather than relying solely on molecular chaperones to refold proteins, this hormetic response causally requires autophagic clearance of misfolded and aggregation-prone proteins to maintain organismal proteostasis. Mechanism: Transient sub-lethal stress triggers HSF-1 trimerization and nuclear translocation alongside calcineurin- and mTORC1-regulated nuclear entry of TFEB (HLH-30 in C. elegans). Together, HSF-1 and TFEB/HLH-30 drive coordinated transcription of core autophagy-related genes (such as ATG8/LC3/lgg-1, ULK1/unc-51, and SQSTM1/p62/sqst-1) and lysosomal hydrolases, increasing autophagic flux to encapsulate and degrade ubiquitinated protein oligomers before they form insoluble toxic aggregates. Prediction: Genetic ablation or RNAi knockdown of TFEB/HLH-30 or core autophagy execution genes (such as ATG5, ATG7, or BECN1/bec-1) specifically after mild stress exposure will abolish both the clearance of polyglutamine or amyloid aggregates and the lifespan extension, even when HSF-1-dependent chaperone induction (HSP70/HSP90) and 26S proteasome activity remain intact.In Caenorhabditis elegans, inhibition of mitochondrial respiration activates an intestinal ELT-2-dependent transcriptional program that increases lysosomal proteolysis during ageing, largely independently of the mitochondrial unfolded protein response. Direct promoter binding by ELT-2, requiring residue R249, and the lysosomal proteases CPR-5 and CPR-8 are necessary for toxic polyglutamine aggregate clearance and mitochondrial stress-induced lifespan extension.GPT-5.6 Sol 2; Claude Opus 5.5 2; Grok 4.7 3noyesno10.1126/sciadv.aeh0657
39Claude Opus 5.5R42630044H2What mechanism limits the anabolic response of aged bone to mechanical loading, and can targeted stimulation restore this response?Aged osteocytes lose mechanosensitivity because the stretch-activated cation channel PIEZO1 is downregulated in cortical bone, so load-induced calcium influx and downstream Wnt1/Wnt10b output collapse; pharmacological agonism of PIEZO1 (e.g. Yoda1) restores the anabolic response without mechanical loading. Mechanism: Reduced PIEZO1 expression/membrane availability in aged osteocytes lowers load-evoked Ca2+ transients, blunting NFAT/YAP-dependent induction of Wnt ligands and suppression of sclerostin, which shifts the formation/resorption balance toward resorption. Direct chemical gating of the residual channel pool bypasses the sensing deficit. Prediction: In aged mice, Yoda1 (or equivalent PIEZO1 agonist) increases cortical thickness and periosteal mineral apposition to a degree comparable with loading in young animals, and this effect is abolished in osteocyte-specific Piezo1 knockouts, whereas overcoming the deficit by increasing strain magnitude alone does not normalize Wnt1/Wnt10b induction.In aged mouse bone, activation of Piezo1 during mechanical loading promotes its membrane co-localization with connexin 43 and increases connexin 43 hemichannel activity through PI3K-Akt signaling. Hemichannel opening increases PGE2 release, suppresses sclerostin, promotes endosteal bone formation, inhibits resorption, and thereby restores the cortical anabolic response to loading.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70687
40Claude Opus 5.5R42664348H5By what causal molecular mechanism does excess adiposity promote osteoarthritis progression, and can disrupting that mechanism reduce joint pathology?Adipose tissue is a major source of a circulating complement-pathway protease that reaches synovial fluid and locally activates the alternative complement cascade on chondrocyte surfaces and damaged matrix, driving cartilage catabolism. Genetic or pharmacological interruption of this adipose-to-joint complement axis should reduce cartilage loss and synovitis in diet-induced obesity without requiring weight loss. Mechanism: Hypertrophic adipocytes secrete a rate-limiting complement activator; it enters the joint, amplifies C3/C5 convertase assembly on cartilage debris, generates C5a and the membrane attack complex sublytically on chondrocytes, which induces MMP-13/ADAMTS-5 and recruits C5aR1+ synovial macrophages. Prediction: Adipocyte-specific deletion of the secreted activator (or C5aR1 blockade) in obese mice reduces OARSI cartilage score and synovitis while body mass, fat mass and joint loading stay unchanged; parabiosis or serum transfer from obese wild-type mice restores pathology in the knockout, and the protection is lost in complement-deficient recipients.Adipocyte-derived extracellular vesicles enriched in mitochondrial components transfer mitochondrial DNA to chondrocytes, where it activates cGAS-STING signaling and causes inflammation, metabolic dysfunction, senescence, and cartilage degeneration. Genetic or pharmacological inhibition of this pathway attenuates cartilage damage, pain behaviors, and gait abnormalities.GPT-5.6 Sol 2; Gemini 4 Argon 1; Grok 4.7 2nonono10.1126/sciadv.aee6780
41Claude Opus 5.5R42665751H2How does age-associated mitochondrial dysfunction initiate and propagate inflammatory signaling that causes ovarian functional decline?Age-associated loss of mitochondrial DNA integrity in oocytes and granulosa cells causes release of mtDNA into the cytosol, which activates the cGAS–STING pathway and initiates type I interferon and NF-κB–driven inflammatory signaling within the follicle. This cell-intrinsic innate-immune activation, rather than infiltrating immune cells, is the initiating event of ovarian functional decline. Mechanism: Oxidative damage and reduced mitophagy in aged follicles permit mtDNA escape through BAX/BAK macropores and VDAC oligomers into the cytosol. Cytosolic mtDNA binds cGAS, generating cGAMP that activates STING, driving TBK1/IRF3 interferon output and NF-κB induction of IL-6, IL-1β and CXCL10. The resulting local inflammatory milieu impairs granulosa steroidogenesis and accelerates follicular atresia. Prediction: Oocyte- or granulosa-specific deletion of Cgas or Sting1, or pharmacological STING inhibition (H-151), in aged or mitochondrially stressed mice preserves follicle reserve, estradiol/AMH levels and fertility while leaving mtDNA copy number and oxidative damage unchanged — i.e. the mitochondrial lesion persists but the functional decline is prevented. Conversely, ovarian expression of a mitochondrial nuclease-resistant mtDNA-releasing mutant (e.g. TFAM haploinsufficiency) reproduces the decline, and it is rescued by STING loss.Ageing-associated mitochondrial DNA leakage from oocytes activates oocyte cGAS, generating cGAMP that triggers STING signaling and passes through gap junctions to activate STING in surrounding granulosa cells. Genetic deletion of oocyte Cgas or pharmacological STING inhibition ameliorates inflammation and ovarian dysfunction, establishing this signaling axis as a causal driver of ovarian ageing.GPT-5.6 Sol 4; Gemini 4 Argon 4; Grok 4.7 4yesnoyes10.1038/s43587-026-01195-y
42Claude Opus 5.5R42689491H3Can in vivo correction of the HGPS-causing mutation rescue molecular, cellular, and structural defects in bone, and how does treatment timing affect efficacy?Mutation correction before the onset of radiographically detectable skeletal disease will prevent bone pathology almost completely, whereas the identical intervention delivered after structural deficits are established will halt progression and partially rebuild bone but will not fully normalize geometry or strength. Efficacy will therefore show a threshold dependence on treatment age rather than a smooth linear decline. Mechanism: Early correction acts on a still-intact progenitor pool and an unremodeled matrix, so normal modeling can proceed. Once progerin has driven progenitor exhaustion/senescence and produced irreversibly altered cortical geometry and hypermineralized or collagen-cross-link-abnormal matrix, correction restores cell function but remodeling cannot re-sculpt macroscopic shape (e.g., clavicular and rib thinning, acro-osteolysis-like resorption) within the animal's lifespan. Prediction: Matched cohorts treated at a pre-symptomatic versus an established-disease age will show equal normalization of molecular markers (progerin levels, osteogenic gene expression) but divergent structural outcomes: near-complete rescue early, and in the late group restoration of dynamic bone formation rate with only partial recovery of cortical area and failure load.Adenine base editing of the pathogenic LMNA variant reduced progerin expression, restored extracellular-matrix deposition and mineralization in progeroid osteoblasts, and partially rescued bone structure and physical properties in mice. In vivo correction was strongly age-dependent, with early postnatal delivery producing substantially greater editing and skeletal benefit than later treatment.GPT-5.6 Sol 4; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70700
43Claude Opus 5.5R42717261H1What mechanism induces pathogenic immune-cell senescence in ulcerative colitis, and does this senescence causally exacerbate the disease?In ulcerative colitis, metabolic stress in mucosal T cells causes mitochondrial DNA to leak into the cytosol, activating the cGAS–STING pathway and driving these cells into a senescent, SASP-secreting state that worsens colitis. Blocking mtDNA release or STING signaling should prevent senescence and attenuate disease. Mechanism: Inflamed mucosal metabolic conditions damage mitochondria in lamina propria T cells; mitochondrial permeability transition/VDAC- or BAX/BAK-dependent pore formation releases mtDNA into the cytosol; cGAS detects it, STING–TBK1–IRF3/NF-κB signaling plus p53/p21 engagement enforces a stable cell-cycle arrest with a pro-inflammatory secretome (IL-6, IL-1β, TNF, chemokines) that recruits and activates neutrophils and macrophages, sustaining epithelial injury. Prediction: In colitis models, T-cell-specific deletion of Cgas or Sting1 (or mtDNA depletion/TFAM-dependent packaging rescue, or inhibition of the mPTP/VDAC oligomerization) reduces p16/p21-positive senescent mucosal T cells and SASP cytokines and lessens histological colitis, whereas deleting the same genes in epithelium or myeloid cells does not reproduce the senescence rescue; cytosolic mtDNA is measurable in sorted UC patient mucosal T cells and correlates with their senescence score.NAD+ metabolic dysregulation causes mitochondrial dysfunction and cytosolic mitochondrial DNA leakage, activating cGAS-STING signaling to drive CD8+ T-cell senescence. These senescent cells exacerbate colitis, while systemic senolytic treatment reduces disease severity; their associated signature also predicts severe disease and non-response to biologic therapy.GPT-5.6 Sol 4; Gemini 4 Argon 4; Grok 4.7 4yesnoyes10.1111/acel.70706
44Claude Opus 5.5R42717261H2What mechanism induces pathogenic immune-cell senescence in ulcerative colitis, and does this senescence causally exacerbate the disease?Senescent immune cells cause resistance to anti-inflammatory therapy in UC: their SASP maintains an inflammatory circuit that is not dependent on the single cytokine being blocked. Adding senescence-targeting treatment restores responsiveness. Mechanism: The SASP is multi-cytokine and self-reinforcing (IL-6/STAT3, IL-1β, TNF, chemokine-driven neutrophil recruitment, and local paracrine spread of senescence to neighboring immune and stromal cells). Blocking one node (TNF, IL-12/23, JAK partially) leaves the rest of the circuit intact, so inflammation persists; removing the senescent source collapses the whole circuit. Prediction: Pre-treatment mucosal senescence burden (p16/p21-high immune cells, SASP transcriptional signature) is higher in future non-responders to anti-TNF and stratifies response better than baseline CRP/histology; in a colitis model refractory to anti-TNF, combining anti-TNF with senolytics or a STING inhibitor restores remission whereas either alone does not.NAD+ metabolic dysregulation causes mitochondrial dysfunction and cytosolic mitochondrial DNA leakage, activating cGAS-STING signaling to drive CD8+ T-cell senescence. These senescent cells exacerbate colitis, while systemic senolytic treatment reduces disease severity; their associated signature also predicts severe disease and non-response to biologic therapy.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1111/acel.70706
45Claude Opus 5.5R42717261H3What mechanism induces pathogenic immune-cell senescence in ulcerative colitis, and does this senescence causally exacerbate the disease?Senescent immune cells are not bystanders but causal drivers of UC severity: their selective removal is sufficient to improve colitis. Senolytic clearance or genetic ablation of p16-high immune cells will reduce inflammation independently of any effect on epithelial cells. Mechanism: A small population of senescent mucosal lymphocytes/myeloid cells acts as a persistent inflammatory hub; because senescent cells are apoptosis-resistant (BCL-2/BCL-xL, PI3K–AKT survival dependence), they accumulate and continuously emit SASP factors that amplify local immune activation and block epithelial regeneration. Eliminating them removes the amplifier. Prediction: In INK-ATTAC/p16-3MR mice (or with dasatinib+quercetin/navitoclax), induced clearance of p16-positive cells after colitis onset lowers colonic SASP cytokines, restores crypt regeneration and reduces disease activity; adoptive transfer of in vitro-senesced T cells into recipients worsens colitis in a dose-dependent manner, while transfer of equal numbers of non-senescent cells does not.NAD+ metabolic dysregulation causes mitochondrial dysfunction and cytosolic mitochondrial DNA leakage, activating cGAS-STING signaling to drive CD8+ T-cell senescence. These senescent cells exacerbate colitis, while systemic senolytic treatment reduces disease severity; their associated signature also predicts severe disease and non-response to biologic therapy.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70706
46Claude Opus 5.5R42723546H5Can a cell-free regenerative treatment alleviate UV-induced skin photoaging, and which causal intracellular mechanism mediates its protective effects?Topical or intradermal application of a cell-free regenerative preparation (mesenchymal stromal cell-derived small extracellular vesicles / conditioned-medium secretome) alleviates UV-induced photoaging primarily by restoring mitophagy in keratinocytes and dermal fibroblasts. Restored PINK1/Parkin-dependent clearance of UV-damaged mitochondria prevents accumulation of depolarized organelles, thereby blocking senescence and sterile inflammation. Mechanism: UV irradiation depolarizes mitochondria and simultaneously blunts autophagic flux, so damaged mitochondria persist. The cell-free preparation delivers/induces PINK1 stabilization and Parkin recruitment, increasing LC3-II-tagged mitophagosome formation and lysosomal delivery; removal of dysfunctional mitochondria lowers mitochondrial ROS, preserves ATP, prevents p16/p21-driven senescence and reduces NF-kB-driven cytokine output, with secondary recovery of collagen I via reduced MMP-1/MMP-3. Prediction: Protection is abolished when mitophagy is blocked — in PINK1- or Parkin-silenced cells, or with Mdivi-1/bafilomycin or mito-QC/mt-Keima reporters showing no flux increase — while generic antioxidant capacity of the preparation is unchanged; conversely mitophagy inducers (urolithin A) reproduce the benefit. Alternatives predicting barrier-first or purely paracrine-growth-factor action would retain protection in PINK1-null skin.Human umbilical cord mesenchymal stem cell secretome protects against UV-induced photoaging by enhancing mitophagy, which clears damaged mitochondria and reduces cytosolic mitochondrial DNA leakage. This suppresses cGAS-STING signaling and downstream inflammatory cytokines; mitophagy inhibition abolishes protection, while STING inhibition restores it.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70701
47Claude Opus 5.5R42725837H2Does the transmembrane protein investigated in this study causally regulate cellular senescence and age-related tissue pathology, and through what signaling mechanism?The transmembrane protein acts as a cell-autonomous driver of senescence: its expression rises in aged and stressed tissue, and raising it is sufficient to push proliferating cells into a stable senescent arrest, while deleting it blocks senescence entry and reduces age-related tissue pathology. Mechanism: Stress-induced upregulation of the protein at the plasma membrane raises its local surface density, enabling ligand-independent clustering and constitutive assembly of an intracellular adaptor complex. The signal converges on NF-kB/p38 to sustain the SASP while p53-p21 and p16-Rb enforce the arrest, creating a feed-forward loop in which SASP cytokines further induce the protein. Prediction: Overexpression in young primary cells with intact p53 produces SA-beta-gal positivity, p21/p16 induction and SASP secretion without added stress, and conditional knockout in an aged or progeroid mouse lowers p16 burden and improves tissue histology and function; a signaling-dead point mutant that still reaches the membrane fails to do either, excluding mere protein-burden or ER-stress effects.PTCHD4 promotes cellular senescence and age-associated tissue dysfunction by enhancing AKT signaling. PTCHD4 deficiency suppressed senescence, alleviated ageing-related phenotypes and bleomycin-induced pulmonary fibrosis in mice, and its anti-senescent effect was reversed by restoring AKT signaling.GPT-5.6 Sol 3; Gemini 4 Argon 2; Grok 4.7 3yesyesno10.1111/acel.70711
48Claude Opus 5.5R42736449H2Can an unannotated protein product that is altered in Alzheimer's disease causally regulate cellular function relevant to disease pathology?A microprotein translated from a small ORF within a lncRNA or 5' uORF is depleted in Alzheimer's disease neurons, and this loss causally impairs mitochondrial respiration independently of any annotated protein at the same locus. Mechanism: The microprotein localizes to the inner mitochondrial membrane and stabilizes assembly of respiratory complex I/IV or ATP synthase dimers. Depletion lowers ATP output and raises mitochondrial ROS, which degrades synaptic maintenance in high-demand neurons. Prediction: CRISPR knockout of only the sORF start codon (leaving the host transcript and any overlapping annotated ORF intact) reduces oxygen consumption rate and complex activity in neurons, and re-expression of the microprotein cDNA from a separate locus rescues it; knockdown of the host lncRNA by itself does not reproduce the defect.A small open reading frame at the MKKS locus encodes a 63-amino-acid microprotein that is the locus's predominant translation product and is downregulated in Alzheimer's disease. Experimental loss of this microprotein impairs mitochondrial respiration in microglia, supporting a causal role in microglial bioenergetics.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1038/s43587-026-01207-x
49Claude Opus 5.5R42749700H2What molecular mechanism links disrupted chromatin regulation to cellular senescence in Hutchinson–Gilford progeria syndrome, and can targeting that mechanism ameliorate disease phenotypes and extend lifespan?Progerin-induced heterochromatin decompaction and nuclear-envelope rupture release genomic and mitochondrial DNA fragments into the cytosol, where cGAS–STING sensing produces the type-I-interferon arm of the SASP; STING loss or inhibition should uncouple nuclear damage from inflammation and improve HGPS phenotypes. Mechanism: Loss of lamin B1/heterochromatin tethering → micronuclei, cytoplasmic chromatin fragments and de-repressed LINE-1 retroelement cDNA → cGAS activation → STING–TBK1–IRF3 → IFN-β and NF-κB SASP → paracrine senescence in vasculature and adipose tissue. Prediction: Cytosolic dsDNA and ISG signatures (Ifit1, Isg15) are elevated in HGPS fibroblasts and G609G aorta and precede p16 induction; Sting1-null G609G mice or STING/TBK1 inhibitor treatment reduce SASP and vascular smooth-muscle loss and extend lifespan, while cGAS-independent DNA-damage signalling (ATM) remains unchanged — separating sensing from damage itself.HMGA1 recruits the deubiquitinase USP7 to stabilize HP1β, which reduces chromatin accessibility at ANGPTL2 and suppresses senescence-associated secretory factors, thereby alleviating HGPS cellular senescence. An HMGA1-derived peptide, UHP, prevents HP1β degradation, reduces senescence, and significantly extends the lifespan of LmnaG609G/G609G mice.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1038/s41467-026-76789-6
50Claude Opus 5.5R42775698H4Can age-associated differences in the human gut microbial community causally reprogram intestinal barrier function, systemic metabolism, and mucosal gene expression, and what mediators contribute to these effects?Transplantation of faecal microbiota from aged human donors into germ-free or antibiotic-depleted young mice is sufficient to cause reduced small-intestinal villus height and surface area, increased paracellular permeability, and a pro-inflammatory mucosal transcriptional programme, whereas microbiota from young donors does not. The effect is donor-age-driven rather than recipient-age-driven, establishing the aged community as an active cause rather than a consequence of host ageing. Mechanism: Age-associated communities are depleted of saccharolytic, butyrate-producing Firmicutes (Faecalibacterium, Roseburia, Eubacterium rectale) and enriched in Proteobacteria/Enterobacteriaceae and pathobionts. Loss of luminal butyrate removes the dominant colonocyte energy substrate and HDAC-inhibitory/HIF-1α-stabilising signal that maintains tight-junction (claudin-1, occludin, ZO-1) expression and epithelial oxygen consumption; the resulting epithelial oxygenation further expands facultative anaerobes, creating a feed-forward loop that degrades barrier integrity and drives NF-κB-dependent mucosal gene expression. Prediction: Recipients of aged-donor microbiota show lower caecal/portal butyrate, reduced claudin/occludin protein, elevated serum FITC-dextran or LPS-binding protein, and villus atrophy; oral tributyrin or butyrate supplementation (or co-colonisation with a butyrogenic consortium) in aged-donor recipients restores villus morphology, tight-junction expression and permeability to young-donor levels without changing overall community membership, while butyrate-receptor-deficient (Ffar2/Ffar3 or Hdac3^ΔIEC) recipients are refractory to that rescue.Transplanting elderly-human gut microbiota into pigs caused reduced ileal villus height, loss of tight-junction proteins, altered plasma metabolites, and coordinated suppression of seven ileal mucosal antiviral-response hub genes compared with young-donor microbiota. The elderly-donor-associated metabolic shift was functionally linked to barrier impairment: Tyr-Phe increased epithelial electrical resistance, whereas prostaglandin E3 decreased it in IPEC-1 cells.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70725
51Claude Opus 5.5R42786310H5Can a commensal microorganism predicted from integrated human ageing profiles causally extend lifespan and healthspan and shift age-associated molecular and microbial states toward younger configurations?A commensal bacterium nominated by multi-omic human ageing profiles (e.g. a Lactobacillus/Lactiplantibacillus- or Akkermansia-class strain whose relative abundance is high in biologically young individuals after adjusting for chronological age) causally extends median and maximum lifespan when colonised in gnotobiotic or conventional mice, and the effect requires live, metabolically active cells. Heat-killed or irradiated preparations of the same strain at matched dose produce no lifespan gain. Mechanism: Viable colonisation sustains continuous production of a secreted small-molecule or peptide effector in the gut lumen that signals to host enterocytes and the enteric immune compartment; killed cells deliver cell-wall ligands but no renewable effector flux, so downstream host signalling is not maintained. Prediction: Live-strain cohorts show a significant (>8%) median lifespan increase versus vehicle, while heat-killed cohorts overlap vehicle survival curves; the lifespan effect scales with faecal colonisation density/CFU and disappears in animals that fail to engraft.Neisseria flavescens was identified as a candidate geroprotective commensal and experimentally shown to extend lifespan and healthspan in Caenorhabditis elegans when administered live. In aged mice, heat-killed N. flavescens shifted the serum metabolome, liver transcriptome and gut microbiome toward younger states, while isolated strains were confirmed to produce predicted beneficial metabolites.GPT-5.6 Sol 2; Gemini 4 Argon 0; Grok 4.7 2nonono10.1038/s43587-026-01220-0
52Claude Opus 5.5R42841227H5Do age-related changes in gut microbial metabolism causally impair antiviral defense during influenza infection, and through what host mechanism can this deficit be counteracted?Ageing reduces gut microbial production of short-chain fatty acids (notably butyrate), and this loss causally impairs antiviral defense in the lung during influenza infection. Restoring butyrate signalling in the host — pharmacologically or by transferring a young/SCFA-producing microbiota — rescues antiviral protection in aged animals. Mechanism: Diminished abundance of fibre-fermenting commensals lowers luminal and systemic butyrate. Butyrate acts on haematopoietic precursors and lung-resident myeloid cells via GPR41/43 and HDAC inhibition, tuning the differentiation of monocytes into patrolling/antiviral phenotypes and limiting damaging neutrophil influx. Without it, aged mice generate excessive tissue-damaging inflammation with weaker type I/III interferon-dependent viral control, raising viral load and mortality. Prediction: Faecal transfer from young to aged mice, or oral butyrate/tributyrin supplementation starting before challenge, lowers lung viral titre and mortality in aged mice; the rescue is abolished in Ffar2/Ffar3-deficient hosts or by myeloid-specific blockade, and germ-free mice colonised with aged microbiota are protected by butyrate but not by fibre alone.Age-associated gut microbiota dysbiosis reduces microbial acetate-production capacity during influenza infection, while acetate supplementation suppresses viral replication and inflammation in senescent human lung fibroblasts. This protection is associated with FFAR2/FFAR3 signaling and increased histone H3 acetylation, supporting acetate as a mechanistic mediator of the ageing gut–lung axis.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70750
53Claude Opus 5.5R42842666H3How do the different cellular compartments of the vascular wall causally contribute to distinct features of Hutchinson-Gilford progeria vascular pathology?Smooth muscle cells are the primary driver of progeroid vascular degeneration, and progerin expression restricted to the smooth muscle layer is sufficient to reproduce medial cell loss, wall thinning and adverse remodelling even when endothelium, fibroblasts and immune cells carry no mutation. Conversely, sparing smooth muscle cells from progerin largely protects the vessel from this degenerative component. Mechanism: Progerin accumulates in the stiff, mechanically loaded medial smooth muscle compartment, where repeated contractile cycling and nuclear strain amplify lamina damage, DNA-damage signalling and mechanosensitive apoptosis; the resulting cell-autonomous attrition cannot be compensated because medial smooth muscle cells have very low replicative reserve. Prediction: In cell-type-restricted progerin models (or three-layer engineered vessels assembled from mutant versus isogenic corrected cells), smooth-muscle-only progerin yields the full medial depletion/wall-thinning phenotype, whereas endothelium-only or fibroblast-only progerin yields near-normal medial cellularity; mechanical unloading or reduced contractile cycling attenuates loss in the smooth-muscle-mutant condition.In trilayer engineered blood vessels, HGPS fibroblasts causally promoted extracellular-matrix dysregulation and fibrotic signaling, while HGPS smooth muscle cells drove collagen accumulation and endothelial cells drove inflammation. Correcting the HGPS-associated mutation by base editing restored many pathological characteristics toward healthy levels.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1126/sciadv.aeh3785
54Claude Opus 5.5R42844664H4Through what causal mechanism does time-restricted feeding protect against accelerated cardiovascular ageing?Time-restricted feeding protects arteries by suppressing NLRP3-inflammasome-driven gasdermin-D pore formation and pyroptosis in endothelial and perivascular myeloid cells; inflammasome activation is the necessary mediator of the accelerated vascular ageing that TRF prevents. Mechanism: The prolonged daily fast lowers postprandial glycemic excursions, lipotoxic DAMPs and mtROS, raises β-hydroxybutyrate (a direct NLRP3 inhibitor) and induces mitophagy, so ASC-speck assembly, caspase-1 activation and IL-1β/IL-18 release fall; less gasdermin-D pore formation preserves endothelial junction integrity, NO bioavailability and prevents the IL-1β→smooth-muscle osteogenic/fibrotic program that stiffens the wall. Prediction: TRF's protection of endothelium-dependent relaxation, pulse-wave velocity and cardiac remodeling is abolished (no further benefit) in Nlrp3−/−, Casp1−/− or Gsdmd−/− animals — i.e. TRF and inflammasome deletion are epistatic, not additive — and conversely constitutively active NLRP3 (Nlrp3^D301N) blocks TRF benefit despite unchanged feeding rhythm; vascular ASC specks and plasma IL-18 fall before any change in blood pressure.Time-restricted feeding enriches acetate-producing Limosilactobacillus, and microbiota-derived acetate is necessary and sufficient for its protection against angiotensin II-induced cardiovascular ageing. Acetate activates GPR43, inhibits HDAC3, increases H4K8 acetylation, and suppresses NLRP3-mediated pyroptosis and cellular senescence.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70728
55Claude Opus 5.5R42853938H3What splicing-dependent mechanism enables resistance to CDK4/6 inhibition, and can disrupting it restore therapy-induced senescence?Resistance to CDK4/6 inhibition is enabled by an aberrant splice isoform of a chromatin-modifying enzyme that loses its regulatory domain while retaining catalytic activity, and this isoform silences the senescence transcriptional program. Forcing expression of the full-length isoform, or blocking production of the truncated one, restores palbociclib-induced senescence. Mechanism: A splicing factor whose expression rises under prolonged CDK4/6 inhibition promotes use of an alternative exon/termination site in a histone methyltransferase or demethylase transcript. The resulting protein is mistargeted to CDKN1A/CDKN2A and SASP loci, where it deposits repressive marks that prevent the stable heterochromatin-plus-p21 state needed for arrest. Cells therefore resume cycling despite sustained RB-pathway inhibition. Prediction: Isoform-specific knockdown of the truncated transcript (antisense oligonucleotide or splice-switching ASO) with no change in total gene mRNA restores SA-β-gal positivity, p21 induction and durable arrest in resistant cells, while catalytically dead rescue of the truncated isoform does not; ChIP-seq shows loss of the repressive mark specifically at senescence loci.Increased inclusion of exon 2 drives SUV39H2 overexpression and CDK4/6 inhibitor resistance. Suppressing SUV39H2 expression or methyltransferase activity removes H3K9me3 from enhancer regions, opens associated promoters, activates p21 and other senescence-associated genes, and restores therapy-induced senescence.GPT-5.6 Sol 2; Gemini 4 Argon 3; Grok 4.7 3yesyesno10.1126/sciadv.aea2001
56Claude Opus 5.5R42723144H4Can naturally occurring inherited genetic variation alter the rate of ovarian ageing on equivalent heterogeneous nuclear backgrounds, and what cellular mechanism mediates this effect?Naturally occurring mitochondrial DNA (mtDNA) haplotype variation is sufficient to shift the rate of ovarian ageing when nuclear background is held equivalent (e.g. conplastic strains or reciprocal F1/outbred crosses sharing a heterogeneous nuclear genome but differing in maternally inherited mtDNA). The effect is mediated by haplotype-specific OXPHOS coupling efficiency in the oocyte–granulosa unit, which sets the rate at which the non-renewable primordial follicle pool is activated and lost. Mechanism: Non-synonymous and tRNA/rRNA variants distinguishing wild-derived mtDNA haplotypes alter complex I/III assembly and proton-leak-adjusted coupling efficiency. Lower coupling raises the ATP/ADP-sensing load on primordial oocytes, de-repressing the PI3K–AKT–FOXO3a/mTORC1 switch that holds follicles dormant, so a larger fraction of the reserve is recruited per cycle and atresia-driven depletion accelerates. The nuclear genome is permissive but not causal because it is identical in distribution across the compared groups. Prediction: At matched chronological age (e.g. 9–12 months), mtDNA-divergent animals on the same heterogeneous nuclear background differ ≥2-fold in primordial follicle counts and in age at cycle irregularity, with the direction predicted by oocyte respirometry (coupling efficiency measured in isolated cumulus–oocyte complexes) and by nuclear-pFOXO3a exit in primordial oocytes — while nuclear SNP association in the same cohort explains no additional variance. Cytoplasmic transfer or mitochondrial replacement reverses the phenotype; a nuclear-background-matched but mtDNA-identical control shows no difference.The OKC-HETW mitochondrial haplotype accelerated ovarian ageing, causing faster primordial-follicle loss, inflammatory and fibrotic remodelling, mitochondrial dysfunction and impaired ovulation relative to the OKC-HETB haplotype on equivalently distributed nuclear backgrounds. Despite normal TFAM expression and elevated total TFAM protein, OKC-HETW ovaries had reduced mitochondrial TFAM, TFAM–mtDNA binding and TOMM20, supporting defective mitochondrial protein import and compromised mtDNA maintenance as the mechanism linking haplotype to accelerated decline.GPT-5.6 Sol 3; Gemini 4 Argon 2; Grok 4.7 2noyesno10.1111/acel.70710
57Claude Opus 5.5R42808804H3What causal mechanism links cochlear ageing to loss of blood-labyrinth barrier integrity and auditory dysfunction?Age-dependent loss of pericyte coverage in the cochlear stria vascularis is the primary causal event driving blood-labyrinth barrier (BLB) leakage and endocochlear potential decline. Pericyte dropout removes the paracrine signals that maintain endothelial tight junctions, so capillaries become permeable before hair cells die. Mechanism: Senescent/stressed cochlear pericytes detach and are lost, reducing pericyte-derived signalling (e.g. PDGFB–PDGFRB axis, angiopoietin-1/TIE2, TGF-beta) to strial endothelium. Endothelial tight-junction and adherens proteins (claudin-5, occludin, ZO-1, VE-cadherin) are downregulated and redistributed, transcytosis rises, plasma proteins and ions enter the intrastrial fluid space, K+ recycling by marginal cells fails and the endocochlear potential falls, raising auditory thresholds. Prediction: In aged animals, quantitative pericyte coverage of strial capillaries falls before measurable tracer leakage and before outer-hair-cell loss; conditional pericyte ablation in young animals reproduces leakage, endocochlear-potential drop and threshold shifts, while pericyte-targeted rescue (PDGF-BB or TIE2 agonism) restores claudin-5, barrier tightness and hearing in aged animals without changing hair-cell counts.Age-dependent loss of IGF-1 lowers SLC25A33 expression in strial pericytes, causing mitochondrial reactive oxygen species accumulation and activation of TGF-β/Smad2 signaling. This drives pericyte phenotypic switching, disrupts blood-labyrinth barrier integrity, and worsens hearing loss, whereas IGF-1 supplementation or SLC25A33 overexpression suppresses the pathway and partially restores auditory function.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1111/acel.70712
58Claude Opus 5.5R42669067H5How does SIRT1 regulate L1 retrotransposon activity, and does this regulation affect cellular senescence?SIRT1 is a direct repressor of L1 retrotransposons: it is recruited to L1 5'UTR promoter regions and deacetylates histone H4K16 (and H3K9/H3K56) there, maintaining a closed chromatin state. Loss or decline of SIRT1 during ageing de-represses L1 transcription, and this de-repression is itself sufficient to accelerate the onset of cellular senescence. Mechanism: SIRT1 occupies the L1 5'UTR, removes H4K16ac, and enables SUV39H1/HP1-dependent H3K9 methylation and heterochromatin compaction over young, full-length L1 loci. Reduced SIRT1 activity (lower NAD+, SIRT1 protein loss, or its known relocalization away from repeats after DNA damage) leaves H4K16ac high, opens the promoter, and allows RNA Pol II to transcribe L1, generating ORF1p/ORF2p and cytoplasmic L1 cDNA. Prediction: SIRT1 ChIP shows enrichment at L1 5'UTRs in proliferating cells that is lost in senescent cells; SIRT1 knockdown raises L1 5'UTR H4K16ac and L1 RNA/ORF1p and triggers premature senescence markers (SA-β-gal, p16, p21), while SIRT1 overexpression or a deacetylase-dependent rescue (not the H363Y catalytic mutant) restores silencing and delays senescence. Critically, blocking L1 downstream of SIRT1 loss — L1 ASOs, ORF2p-targeting shRNA, or reverse-transcriptase inhibitors such as lamivudine — prevents the senescence acceleration without restoring SIRT1 activity, placing L1 causally downstream.SIRT1 suppresses L1 transcription and retrotransposition by promoting the association and recruitment of Lamin B1 and KAP1 at the L1 5′-UTR, increasing repressive H3K9me3 chromatin. SIRT1 deficiency derepresses L1, activates cGAS–STING signaling, and induces cellular senescence; reverse-transcriptase inhibition with 3TC rescues this phenotype.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70689
59Claude Opus 5.5R42806003H5What intercellular mechanism causes placental senescence during maternal ageing, and can disrupting this mechanism restore placental and offspring health?Maternal ageing drives placental senescence through transfer of senescence-promoting signals from aged maternal decidual cells to fetal trophoblast across the maternal–fetal interface. Blocking this directional paracrine transfer in aged mothers prevents trophoblast senescence and restores fetal growth. Mechanism: Aged decidual stromal and immune cells acquire a SASP enriched in IL-6, IL-1β, CXCL ligands and TGF-β-family factors. These diffuse into the adjacent trophoblast layer, where receptor-driven NF-κB and p38 signalling induces p16/p21 and a secondary senescence programme — paracrine 'bystander' senescence spreading from the maternal to the fetal compartment. The senescent trophoblast loses nutrient-transport and endocrine capacity, causing growth restriction. Prediction: In aged-mother/young-decidua chimeric or decidua-specific interventions (e.g. decidual-restricted p16-INK-ATTAC clearance, or decidua-restricted IL-1R/IL-6R knockout), fetal trophoblast senescence markers and placental efficiency normalise, whereas the same intervention applied to trophoblast alone gives partial rescue. Spatial transcriptomics should show a senescence gradient decaying with distance from the decidual border, with maternal-compartment onset preceding fetal-compartment onset in time-course sampling.Early inflammation and senescence-associated secretory phenotype activity induce IRF5-dependent CD38 expression in macrophages, which depletes NAD+ in decidual stromal cells and thereby promotes age-related placental senescence. Genetic or pharmacological disruption of this axis, including CD38 inhibition or NAD+ precursor treatment, reduces placental senescence, rescues intrauterine growth restriction, and improves offspring metabolic outcomes.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1038/s41467-026-77217-5
60Claude Opus 5.5R42748238H3What molecular mechanism causes age-related vascular deterioration to promote systemic senescence and shorten lifespan?Aged endothelial cells lose a key metabolic enzyme that supplies a circulating NAD+ precursor to downstream tissues, so systemic NAD+ availability falls and senescence spreads organism-wide. Restoring the endothelial enzyme, or supplying the precursor, reverses systemic senescence and extends lifespan. Mechanism: Microvascular endothelium normally secretes an extracellular NAD+-biosynthetic enzyme (e.g., eNAMPT in extracellular vesicles) into plasma. Age-related endothelial dysfunction and capillary rarefaction reduce this secretion; parenchymal cells in muscle, brain, heart and kidney consequently cannot maintain NAD+ pools, sirtuin activity drops, mitochondrial and DNA-repair capacity fail, and cells enter a p16/p21 senescent state with SASP output that further damages the vasculature — a feed-forward loop. Prediction: Endothelial-specific knockout of the secreted enzyme in young mice lowers plasma precursor levels, drops NAD+ and raises senescence markers in distal non-vascular organs, and shortens lifespan; endothelial-specific overexpression (or precursor repletion) in aged mice restores distal-tissue NAD+, clears senescence and extends lifespan. Parenchymal-cell-specific knockout of the same enzyme does not reproduce the systemic phenotype, distinguishing a vascular-origin secreted factor from cell-autonomous metabolic decline.Progressive activation of endothelial C/EBPβ induces AEP, which cleaves NAMPT and thereby causes systemic NAD+ depletion, vascular dysfunction, senescence, and reduced lifespan. Genetic disruption of AEP or expression of cleavage-resistant NAMPT N136A ameliorates these effects, while AEP inhibition or NMN supplementation alleviates age-related vascular decline.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1126/sciadv.aed1961

Hypotheses from the repeat generation of Claude Opus 5.5

The repeat generation, with tool use confirmed to be disabled, belongs to the sensitivity analysis (Sections 2.9 and 3.1 of the manuscript). Selection by the same rule retained 17 hypotheses, 1 of them with a borderline decision.

No.▲Generator model▲Task▲Hypothesis▲Research question▲Full text of the hypothesis▲Reference finding of the article▲Scores of the three judges from other companies▲Match at the primary threshold▲Borderline majority decision▲Match at the strict threshold▲Article DOI▲
1Claude Opus 5.5, repeat generationR42601407H4Through what molecular mechanism does physical activity delay ovarian ageing and extend reproductive lifespan?Physical activity delays ovarian ageing primarily by restoring NAD+ availability in oocytes and granulosa cells, which reactivates SIRT1/SIRT3-dependent mitochondrial quality control. Mechanism: Contraction-driven energy turnover raises NAMPT expression and flux through the NAD+ salvage pathway in systemic circulation and ovarian tissue, countering age-related NAD+ decline. Higher NAD+ raises SIRT1 and SIRT3 deacetylase activity, increasing PGC-1α-driven biogenesis, SOD2 activation and mitophagy. Oocyte mitochondrial membrane potential and ATP for meiotic spindle assembly are preserved, lowering aneuploidy and atresia rates. Prediction: Ovary-specific Nampt deletion, or granulosa/oocyte-specific Sirt3 deletion, abolishes exercise-associated gains in oocyte ATP, spindle normality and litter size in aged mice, while sedentary aged mice given NMN partially reproduce the exercise phenotype without further additive benefit from exercise.Physical activity delays ovarian ageing in mice partly by increasing ovarian adiponectin signaling. Its protective effects were attenuated in adiponectin-deficient mice, while pharmacological activation of adiponectin receptors with AdipoRon delayed ovarian ageing and extended reproductive lifespan.GPT-5.6 Sol 2; Gemini 4 Argon 1; Grok 4.7 2nonono10.1038/s43587-026-01177-0
2Claude Opus 5.5, repeat generationR42605193H1Which circulating regulator causally promotes cellular senescence and organismal ageing, and through what intracellular signaling mechanism does it act?Rising circulating interleukin-11 (IL-11) from aged adipose and stromal cells is a causal driver of cellular senescence and shortened healthspan, not a downstream marker of inflammation. IL-11 acts on IL11RA1/gp130 on fibroblasts, hepatocytes and muscle progenitors to establish a self-sustaining senescent, fibrotic state. Mechanism: IL-11 binding to IL11RA1/gp130 activates JAK1/STAT3 and, non-canonically, ERK-dependent stabilization of translation regulators; STAT3 drives CDKN1A and IL11 itself, forming an autocrine loop, while ERK/mTORC1 output blocks autophagic clearance of damaged mitochondria. The resulting ROS and cGAS-independent chronic STAT3 tone lock cells out of cycle and amplify SASP secretion, which spreads senescence to neighbouring cells. Prediction: Neutralizing IL-11 or deleting Il11ra1 only in non-immune parenchymal cells of 18-month-old mice should reduce p16/p21-positive cell burden in liver, muscle and kidney within 8 weeks and extend remaining lifespan, whereas IL-6 blockade in matched animals reduces circulating SASP cytokines without lowering p16 burden. Conversely, chronic low-dose recombinant IL-11 in young mice should induce tissue senescence that is abolished by concurrent JAK1 inhibition but not by NF-kB inhibition.ANGPTL8 directly interacts with AKT2 and activates the AKT–mTOR–S6K pathway, thereby driving cell-autonomous adipocyte senescence. Deleting Angptl8 in mice reduces adipose senescence, delays age-related functional decline, and extends lifespan, while genetic or pharmacological pathway inhibition abolishes ANGPTL8-induced senescence.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1111/acel.70671
3Claude Opus 5.5, repeat generationR42608398H4Can a microbiota-derived factor directly cause the cellular dysfunction and progressive tissue remodeling characteristic of idiopathic pulmonary fibrosis, and if so, through what mechanism?A specific bacterial peptide released by lung-resident staphylococci (a fragment of a bacterial cell-wall transglycosylase, 'corisin-like') is sufficient on its own to cause alveolar type II epithelial cell apoptosis and to convert a self-limited lung injury into progressive fibrosis. Mechanism: The peptide inserts into the epithelial plasma membrane and triggers intrinsic mitochondrial apoptosis (BAX/BAK activation, cytochrome c release, caspase-9/3 cleavage) in AT2 cells. Loss of AT2 cells removes the alveolar progenitor pool needed for re-epithelialization, and the dying cells release TGF-β and DAMPs that activate neighboring fibroblasts, so each microbial bloom produces a new wave of injury and matrix deposition. Prediction: Intratracheal administration of the synthetic peptide alone to mice with mild, otherwise resolving bleomycin injury produces sustained increases in collagen and AT2 apoptosis, while a single-residue-mutant inactive analogue does not; neutralizing monoclonal antibody against the peptide, or colonization with an isogenic mutant unable to generate it, blocks fibrosis progression despite equal bacterial burden.The microbiota-derived peptide corisin directly disrupts epithelial proteostasis by targeting and enhancing the ubiquitin-proteasome system, thereby inducing apoptosis, cellular senescence, epithelial-mesenchymal transition, and fibrotic remodeling. Depleting native corisin from patient bronchoalveolar lavage fluid removes its proapoptotic activity, while constitutive corisin expression in mice causes spontaneous progressive pulmonary fibrosis and worsens bleomycin-induced injury and mortality.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1038/s41467-026-76162-7
4Claude Opus 5.5, repeat generationR42630044H2What mechanism limits the anabolic response of aged bone to mechanical loading, and can targeted stimulation restore this response?Diminished anabolic response in aged bone results from reduced osteocytic expression and membrane availability of the mechanosensitive cation channel PIEZO1, so the same strain magnitude produces a smaller Ca2+ influx and weaker downstream YAP/TAZ-dependent transcription of Wnt1 and Wnt10b. Pharmacological agonism of the remaining channels with Yoda1 (or equivalent chemical activation) restores load-induced cortical bone formation in aged animals. Mechanism: Age-related decline in PIEZO1 transcript/protein and altered membrane lipid order reduce strain-gated Ca2+ entry in osteocytes; loss of Ca2+-dependent YAP/TAZ nuclear translocation lowers Wnt ligand output, shifting the osteocyte secretome toward sclerostin-high, RANKL-high signalling. Chemical agonists lower the strain threshold for channel opening, re-coupling loading to Wnt-driven periosteal formation. Prediction: In aged mice, in vivo tibial loading plus Yoda1 restores periosteal mineral apposition rate and cortical thickness to young-adult levels, with recovery of osteocytic Wnt1/Wnt10b induction; the rescue is abolished in osteocyte-specific Piezo1 conditional knockouts, whereas young mice gain little additional benefit from the agonist.In aged mouse bone, activation of Piezo1 during mechanical loading promotes its membrane co-localization with connexin 43 and increases connexin 43 hemichannel activity through PI3K-Akt signaling. Hemichannel opening increases PGE2 release, suppresses sclerostin, promotes endosteal bone formation, inhibits resorption, and thereby restores the cortical anabolic response to loading.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70687
5Claude Opus 5.5, repeat generationR42647635H4Does experimentally increasing somatic mitochondrial DNA mutation burden cause premature ageing and shortened lifespan in a large mammal?Elevating the somatic mtDNA point-mutation rate in a large mammal is sufficient to cause systemic premature ageing and shortened lifespan. Specifically, pigs carrying a homozygous proofreading-deficient knock-in allele of the mitochondrial DNA polymerase gamma catalytic subunit (POLG D257A-equivalent) will accumulate mtDNA point mutations at several-fold the wild-type rate and develop multi-organ ageing phenotypes — kyphosis, sarcopenia, alopecia and skin thinning, osteoporosis, anaemia, cardiomyopathy, hearing and visual loss — with a proportionate reduction in median and maximum lifespan relative to littermate controls. Mechanism: Loss of 3'-5' exonuclease proofreading during mtDNA replication raises the fixation rate of base-substitution errors. Mutations accumulate across the 13 OXPHOS-subunit genes and 22 tRNAs, producing mistranslated and misassembled respiratory complexes. Falling complex I/III/IV assembly lowers membrane potential and ATP output, raises NADH/NAD+ ratio, and impairs biosynthesis and repair in tissues with high oxidative demand, so damage outruns maintenance in many organs simultaneously. Prediction: Mutant pigs show a dose-dependent relationship: heterozygotes have an intermediate mtDNA mutation load and intermediate phenotype onset, homozygotes the highest load and earliest onset, with mutation burden measured by duplex or single-molecule sequencing correlating quantitatively (per-animal r on the order of 0.6-0.9) with a composite ageing score and inversely with survival. Crucially, the phenotype must track mutation load rather than any off-target effect of the engineered allele: a catalytically matched control knock-in that alters POLG protein sequence without impairing proofreading fidelity produces neither the mutation accumulation nor the phenotype.Prime-edited pigs expressing proofreading-deficient POLG accumulated elevated somatic mitochondrial DNA mutations and developed systemic premature-ageing phenotypes. These included weight loss, rough hair, anaemia, tissue abnormalities, increased apoptosis, elevated senescence-associated markers, and shortened lifespan.GPT-5.6 Sol 4; Gemini 4 Argon 4; Grok 4.7 4yesnoyes10.1126/sciadv.adu0632
6Claude Opus 5.5, repeat generationR42717261H3What mechanism induces pathogenic immune-cell senescence in ulcerative colitis, and does this senescence causally exacerbate the disease?Senescent immune cells are causally required for disease progression in ulcerative colitis: their selective removal is sufficient to attenuate established colitis even without blocking upstream cytokines. Mechanism: Senescent lamina propria immune cells are apoptosis-resistant through BCL-2/BCL-xL and PI3K–AKT dependence, so they persist and continuously secrete SASP factors that degrade epithelial barrier function (claudin loss, goblet-cell depletion) and recruit inflammatory myeloid cells. Removing these cells collapses the SASP source and permits barrier restitution. Prediction: Senolytic treatment (navitoclax or dasatinib+quercetin) or genetic clearance of p16-positive cells begun after colitis is established lowers histological score, restores barrier integrity, and reduces SASP transcripts; transfer of sorted senescent (p21-high) colonic immune cells into recipients worsens colitis, while the same number of non-senescent counterparts does not. Dose-matched broad immunosuppression would reduce inflammation without specifically lowering p16/p21 burden, discriminating the two explanations.NAD+ metabolic dysregulation causes mitochondrial dysfunction and cytosolic mitochondrial DNA leakage, activating cGAS-STING signaling to drive CD8+ T-cell senescence. These senescent cells exacerbate colitis, while systemic senolytic treatment reduces disease severity; their associated signature also predicts severe disease and non-response to biologic therapy.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70706
7Claude Opus 5.5, repeat generationR42723546H5Can a cell-free regenerative treatment alleviate UV-induced skin photoaging, and which causal intracellular mechanism mediates its protective effects?A cell-free secretome/extracellular-vesicle preparation from stem cells alleviates UV photoaging primarily by re-activating PINK1–Parkin-dependent mitophagy in keratinocytes and dermal fibroblasts. Clearance of depolarized mitochondria prevents cytosolic leakage of mitochondrial DNA, which is the proximate danger signal driving cGAS–STING activation, SASP secretion and senescence after chronic UV. Mechanism: Vesicle cargo (PINK1 transcript/protein, Parkin-stabilizing factors, or deubiquitinase-modulating miRNAs) restores recruitment of Parkin to damaged mitochondria, lowers the burden of depolarized organelles, reduces cytosolic mtDNA, and thereby suppresses cGAS–STING–TBK1–IRF3/NF-κB signalling, type I interferon and IL-6/IL-8 release. Prediction: In UV-irradiated skin or fibroblasts, treatment raises LC3/LAMP1–mitochondria colocalization and lowers cytosolic mtDNA, p16/p21 and SA-β-gal; siRNA knockdown of PINK1 (or Parkin) abolishes the anti-senescence and anti-inflammatory benefit, whereas it persists when autophagy of non-mitochondrial cargo is left intact. A STING agonist given downstream of restored mitophagy reinstates the SASP, showing mtDNA–STING is the mediated arm.Human umbilical cord mesenchymal stem cell secretome protects against UV-induced photoaging by enhancing mitophagy, which clears damaged mitochondria and reduces cytosolic mitochondrial DNA leakage. This suppresses cGAS-STING signaling and downstream inflammatory cytokines; mitophagy inhibition abolishes protection, while STING inhibition restores it.GPT-5.6 Sol 4; Gemini 4 Argon 5; Grok 4.7 4yesnoyes10.1111/acel.70701
8Claude Opus 5.5, repeat generationR42749700H5What molecular mechanism links disrupted chromatin regulation to cellular senescence in Hutchinson–Gilford progeria syndrome, and can targeting that mechanism ameliorate disease phenotypes and extend lifespan?In HGPS, progerin accumulation causes a gain of the histone acetyltransferase KAT7 (HBO1) activity at the promoters of senescence effector genes; the resulting excess H3K14 acetylation licenses transcription of CDKN2A/p16INK4a and SASP genes, and KAT7 loss-of-function is therefore sufficient to reverse senescence and extend lifespan in progeroid animals. Mechanism: Progerin-driven heterochromatin disorganization redistributes KAT7/HBO1 to euchromatic senescence loci. Elevated H3K14ac opens these promoters to the general machinery and drives p16INK4a and NF-kB-dependent SASP transcription, locking the cell into a stable senescent, pro-inflammatory state independent of further DNA damage. Prediction: ChIP-seq of HGPS fibroblasts shows KAT7 and H3K14ac enrichment specifically at CDKN2A and core SASP promoters versus isogenic controls; CRISPR knockout or small-molecule inhibition of KAT7 (but not of unrelated HATs such as p300) lowers p16INK4a and IL-6/IL-1B secretion, restores proliferation, and lentiviral/AAV-delivered Kat7 knockdown extends median lifespan of Zmpste24-/- or LmnaG609G mice; H3K14ac rescue by catalytically active KAT7 re-establishes senescence.HMGA1 recruits the deubiquitinase USP7 to stabilize HP1β, which reduces chromatin accessibility at ANGPTL2 and suppresses senescence-associated secretory factors, thereby alleviating HGPS cellular senescence. An HMGA1-derived peptide, UHP, prevents HP1β degradation, reduces senescence, and significantly extends the lifespan of LmnaG609G/G609G mice.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1038/s41467-026-76789-6
9Claude Opus 5.5, repeat generationR42775698H4Can age-associated differences in the human gut microbial community causally reprogram intestinal barrier function, systemic metabolism, and mucosal gene expression, and what mediators contribute to these effects?Germ-free or antibiotic-depleted young mice colonized with faecal microbiota from elderly human donors develop impaired intestinal barrier function (reduced villus height, shallower crypts, increased paracellular permeability) relative to recipients of young-donor microbiota, demonstrating that the aged human community is sufficient to cause host intestinal dysfunction independent of host age. Mechanism: Aged donor communities are depleted of saccharolytic short-chain fatty acid producers and enriched in proteolytic and mucin-degrading taxa; the resulting fall in luminal butyrate starves colonocyte mitochondrial beta-oxidation, raises epithelial oxygenation, and lowers expression of tight-junction and mucin genes, thinning the mucus layer and loosening the junctional seal. Prediction: Aged-donor recipients show lower caecal butyrate, reduced Occludin/Claudin-1/Muc2 transcript and protein levels, higher serum FITC-dextran or LPS-binding protein, and these deficits are rescued by oral butyrate or tributyrin supplementation without altering the transplanted community's composition.Transplanting elderly-human gut microbiota into pigs caused reduced ileal villus height, loss of tight-junction proteins, altered plasma metabolites, and coordinated suppression of seven ileal mucosal antiviral-response hub genes compared with young-donor microbiota. The elderly-donor-associated metabolic shift was functionally linked to barrier impairment: Tyr-Phe increased epithelial electrical resistance, whereas prostaglandin E3 decreased it in IPEC-1 cells.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70725
10Claude Opus 5.5, repeat generationR42786310H4Can a commensal microorganism predicted from integrated human ageing profiles causally extend lifespan and healthspan and shift age-associated molecular and microbial states toward younger configurations?The commensal shifts the host transcriptome, DNA-methylation clock and gut metagenome of aged animals toward younger configurations, i.e. the intervention produces a coordinated rejuvenation signature rather than isolated phenotypic improvement. Mechanism: Reduced inflammatory tone plus metabolite-driven epigenetic substrate changes (acetyl-CoA, SAM, alpha-ketoglutarate availability) remodel chromatin and transcription in liver, intestine and immune cells, while niche competition and cross-feeding displace age-expanded pathobionts (Enterobacteriaceae, Desulfovibrio) and restore butyrate producers. Prediction: Treated old animals show significantly reduced epigenetic-clock age acceleration and transcriptomic age versus age-matched controls, with the direction of change in old-vs-young differential genes reversed (negative enrichment correlation), and metagenomic composition moving along the young-old principal axis toward young centroid; effect sizes correlate across host and microbial readouts within individuals.Neisseria flavescens was identified as a candidate geroprotective commensal and experimentally shown to extend lifespan and healthspan in Caenorhabditis elegans when administered live. In aged mice, heat-killed N. flavescens shifted the serum metabolome, liver transcriptome and gut microbiome toward younger states, while isolated strains were confirmed to produce predicted beneficial metabolites.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1038/s43587-026-01220-0
11Claude Opus 5.5, repeat generationR42821663H3Through what causal mechanism does hypertension accelerate osteoarthritis in joints predisposed by aging or mechanical injury?Hypertension accelerates osteoarthritis because circulating and locally generated angiotensin II acts on chondrocyte and synovial AT1 receptors (AGTR1). This drives catabolic signalling and chondrocyte senescence that add to the damage already present after injury or in the aged joint. Mechanism: Angiotensin II binds AT1R on chondrocytes, synovial fibroblasts and subchondral osteoclast precursors. This activates NADPH oxidase and ROS production, which induces NF-κB/MAPK-dependent expression of MMP13, ADAMTS5 and IL-6/IL-1β, together with p16/p21-mediated senescence. The joint renin-angiotensin system is itself upregulated by mechanical injury and aging, so systemic hypertension raises a ligand whose receptor the injured joint has already sensitized. Prediction: In destabilization of the medial meniscus (DMM) or aged mice, angiotensin II infusion worsens OARSI scores compared with normotensive controls. Chondrocyte-specific Agtr1a deletion or losartan abolishes this effect. Lowering blood pressure to the same degree with hydralazine, which does not block AT1R, rescues joint damage much less.Hypertension-associated circulating arginine vasopressin activates up-regulated AVPR1A in osteoarthritis-primed chondrocytes, driving NR4A3-dependent catabolic signaling and suppressing anabolic regulation. Genetic deletion or pharmacological inhibition of AVPR1A prevents the hypertension-induced acceleration of osteoarthritis.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1126/science.aed8207
12Claude Opus 5.5, repeat generationR42822442H4How does nutrient scarcity in the solid-tumor microenvironment cause senescence and loss of persistence in adoptively transferred antitumor immune cells?Glucose scarcity in the tumor microenvironment drives senescence in adoptively transferred T cells through a cell-intrinsic energy-stress kinase relay: falling ATP/AMP ratio activates AMPK, which engages the scaffold TAB1 to autoactivate p38 MAPK, installing a stable p21/p16-dependent senescence program rather than reversible exhaustion. Disabling this relay (AMPK or TAB1 or p38 loss) preserves persistence without restoring glucose. Mechanism: Low glucose → reduced glycolytic ATP → AMP-bound AMPK → AMPK-dependent TAB1 association with p38α → MAPK-independent (non-MKK) p38 autophosphorylation → p53/p21 and p16 induction, SASP, permanent cell-cycle withdrawal and effector-function loss. Prediction: In CAR-T or TCR-T cells transferred into glucose-poor tumors, TAB1 knockout or a TAB1-p38 interaction-blocking mutant abolishes SA-β-gal/p16/p21 induction and restores intratumoral expansion and tumor control, whereas it has no effect in glucose-replete subcutaneous or in vitro conditions; pharmacologic AMPK activation in high glucose reproduces senescence.Glucose deprivation activates AMPK in transferred Th9 cells, initiating a ZAK–NIK cascade that suppresses noncanonical NF-κB signaling and IL-9 production, thereby driving cellular senescence. Restoring glucose or targeting ZAK reduces senescence, improves Th9-cell persistence, and enhances CAR-Th9 efficacy against solid tumors.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1016/j.immuni.2026.09.004
13Claude Opus 5.5, repeat generationR42858183H3Can a defined intervention preserve lysosomal function and protein homeostasis during aging, thereby extending lifespan and maintaining organismal function, and through what mechanism?Preventing lysosomal lipofuscin and oxidized-lipid accumulation by enhancing lysosomal lipid catabolism (lysosomal acid lipase or LIPL-4 induction) preserves hydrolase function during aging and extends lifespan through a lipid-messenger signal rather than bulk clearance alone. Mechanism: Undegraded lipid and lipofuscin bind hydrolases and quench their activity, progressively poisoning the lysosome. Enhanced lysosomal lipolysis clears this load and releases specific fatty-acid ligands that engage nuclear receptors, which in turn reinforce lysosomal and mitochondrial gene programs. Prediction: Lysosomal lipase induction reduces autofluorescent lipofuscin and restores cathepsin activity in aged tissue, while a catalytically dead enzyme does not. Supplying the specific released fatty acid to animals lacking the lipase partially restores lifespan extension, showing a signaling rather than purely clearance mechanism.20(S)-ginsenoside Rg3 extended lifespan and preserved physiological and neuronal function in Caenorhabditis elegans by restoring lysosomal activity and autophagic flux. Rg3 remodeled neuronal lysosomal phosphoinositide signatures toward increased PI(4)P and reduced PI(3)P, suppressed RSKS-1/S6K–TORC1 signaling, and promoted nuclear HLH-30/TFEB activity, thereby improving proteostasis and resistance to polyglutamine- and α-synuclein-associated toxicity.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1111/acel.70755
14Claude Opus 5.5, repeat generationR42723144H4Can naturally occurring inherited genetic variation alter the rate of ovarian ageing on equivalent heterogeneous nuclear backgrounds, and what cellular mechanism mediates this effect?Naturally occurring mitochondrial DNA (mtDNA) haplotype variation is sufficient to change the rate of ovarian ageing when nuclear genomes are equivalently heterogeneous: conplastic/mitochondrial-nuclear-exchange females carrying a divergent wild-derived mtDNA haplotype on a randomized heterogeneous nuclear background will deplete the primordial follicle reserve faster (or slower) than haplotype-matched siblings. The mediating cellular defect is reduced OXPHOS coupling efficiency in the oocyte-granulosa unit, which raises per-follicle oxidative burden and atresia probability. Mechanism: Haplotype-specific coding and regulatory substitutions in mtDNA-encoded complex I/III/IV subunits alter electron-transfer kinetics and proton leak. In the metabolically quiescent but long-lived primordial and primary oocyte, even a small decrease in P/O ratio chronically elevates superoxide production at complexes I and III, oxidizing mtDNA, cardiolipin and the meiotic spindle machinery. Damaged follicles cross an atresia threshold earlier, so the exponential decay constant of the resting pool increases while the starting pool size (set at birth) is unchanged. Prediction: At 2-3 months of age follicle counts and litter size are indistinguishable between haplotypes, but the log-linear decay slope of primordial follicle number from 3 to 12 months differs significantly between mtDNA haplotypes on the same heterogeneous nuclear background; isolated oocytes/cumulus complexes from the fast-ageing haplotype show lower ATP-linked respiration, higher leak-fraction respiration and higher mitochondrial superoxide (MitoSOX) before follicle loss is detectable. A nuclear-genetics-only explanation predicts no haplotype effect after nuclear randomization; a purely maternal-environment explanation predicts the effect disappears after embryo transfer or cross-fostering, which it should not.The OKC-HETW mitochondrial haplotype accelerated ovarian ageing, causing faster primordial-follicle loss, inflammatory and fibrotic remodelling, mitochondrial dysfunction and impaired ovulation relative to the OKC-HETB haplotype on equivalently distributed nuclear backgrounds. Despite normal TFAM expression and elevated total TFAM protein, OKC-HETW ovaries had reduced mitochondrial TFAM, TFAM–mtDNA binding and TOMM20, supporting defective mitochondrial protein import and compromised mtDNA maintenance as the mechanism linking haplotype to accelerated decline.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 3yesnono10.1111/acel.70710
15Claude Opus 5.5, repeat generationR42585340H3Which molecular mechanism enables mild cellular stress to preserve protein homeostasis during ageing and extend organismal lifespan?Mild cellular stress activates TFEB/HLH-30, which induces genes for autophagy and lysosome formation. The resulting increase in autophagic flux removes protein aggregates and is required for the lifespan extension. Mechanism: Stress inhibits mTORC1, which leads to TFEB dephosphorylation and nuclear entry. TFEB then activates CLEAR-motif genes covering autophagosome formation, lysosomal acidification and hydrolases. Greater delivery of aggregates to lysosomes lowers the proteotoxic load in ageing cells. Prediction: Mutants lacking HLH-30, or with autophagy genes such as bec-1 or atg-18 knocked down, should show neither better aggregate clearance nor longer life after mild stress. Autophagic flux reporters should rise only when HLH-30 is present.In Caenorhabditis elegans, inhibition of mitochondrial respiration activates an intestinal ELT-2-dependent transcriptional program that increases lysosomal proteolysis during ageing, largely independently of the mitochondrial unfolded protein response. Direct promoter binding by ELT-2, requiring residue R249, and the lysosomal proteases CPR-5 and CPR-8 are necessary for toxic polyglutamine aggregate clearance and mitochondrial stress-induced lifespan extension.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1126/sciadv.aeh0657
16Claude Opus 5.5, repeat generationR42669067H5How does SIRT1 regulate L1 retrotransposon activity, and does this regulation affect cellular senescence?SIRT1 is a direct, locus-resident silencer of young L1 elements: it is recruited to the L1 5'UTR promoter and keeps it in a deacetylated, heterochromatic state. Age- or stress-associated loss of SIRT1 from these loci de-represses L1 transcription, and this de-repression is a causal driver of senescence entry rather than a passive consequence of it. Mechanism: SIRT1 occupies the CpG-island promoter in the 5'UTR of evolutionarily young (L1HS/L1PA) elements and removes activating acetyl marks (H4K16ac, H3K9ac) from local nucleosomes. Deacetylated H3K9 becomes a substrate for SUV39H1/SETDB1-mediated H3K9 trimethylation and HP1 binding, locking the promoter in heterochromatin. When SIRT1 protein or NAD+ falls, H4K16ac/H3K9ac accumulate, H3K9me3 and HP1 are lost, and RNA Pol II engages the L1 promoter. Prediction: ChIP of SIRT1 in proliferating fibroblasts shows enrichment at L1 5'UTRs that is lost during replicative or oxidative senescence; acute SIRT1 knockdown raises H4K16ac and lowers H3K9me3 specifically at L1 5'UTRs within 48 h, before SASP gene induction, and catalytically dead SIRT1 (H363Y) fails to restore silencing while wild-type SIRT1 does. Crucially, the chromatin change at L1 must precede, not follow, p16/IL-6 induction in a time-course.SIRT1 suppresses L1 transcription and retrotransposition by promoting the association and recruitment of Lamin B1 and KAP1 at the L1 5′-UTR, increasing repressive H3K9me3 chromatin. SIRT1 deficiency derepresses L1, activates cGAS–STING signaling, and induces cellular senescence; reverse-transcriptase inhibition with 3TC rescues this phenotype.GPT-5.6 Sol 3; Gemini 4 Argon 3; Grok 4.7 2yesyesno10.1111/acel.70689
17Claude Opus 5.5, repeat generationR42806003H5What intercellular mechanism causes placental senescence during maternal ageing, and can disrupting this mechanism restore placental and offspring health?Maternal ageing drives accumulation of senescent maternal decidual cells whose SASP acts across the maternal-fetal interface to induce paracrine senescence in fetal-derived trophoblast, and blocking this specific SASP signal restores placental function and offspring growth. The decidual compartment is the initiating site; trophoblast senescence is secondary and propagated, not autonomous. Mechanism: Age-associated metabolic stress and DNA damage in decidual stromal cells activate cGAS-STING and NF-kB, producing a SASP enriched in IL-6, IL-1b, CXCL1 and TGF-b. These factors diffuse into the adjacent trophoblast layer, imposing a bystander senescence program (p16/p21, SA-b-gal, loss of proliferation) that reduces labyrinth vascular branching and nutrient transporter density, impairing fetal growth. Prediction: Senolytic or SASP-neutralising intervention restricted to the maternal compartment (or maternal-genotype-specific deletion of a SASP driver such as Il6 or Cgas) rescues trophoblast senescence markers, labyrinth vascularisation and fetal weight, whereas the same intervention targeted to fetal trophoblast genotype does not; in young hosts, transfer of aged decidual conditioned medium reproduces trophoblast senescence.Early inflammation and senescence-associated secretory phenotype activity induce IRF5-dependent CD38 expression in macrophages, which depletes NAD+ in decidual stromal cells and thereby promotes age-related placental senescence. Genetic or pharmacological disruption of this axis, including CD38 inhibition or NAD+ precursor treatment, reduces placental senescence, rescues intrauterine growth restriction, and improves offspring metabolic outcomes.GPT-5.6 Sol 2; Gemini 4 Argon 2; Grok 4.7 2nonono10.1038/s41467-026-77217-5

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