Deep networks that estimate ground reaction forces and centers of pressure from motion-capture markers are validated within one laboratory, in a seldom-stated coordinate frame, and sometimes with trial-level splits that put the same person in the training and the test set. We measured what each practice costs, and whether such models transfer between laboratories. Three sequence models (convolutional, convolutional-recurrent, and convolutional-transformer) were evaluated on two independent datasets, locomotion (6 subjects) and occupational patient handling (10 subjects), under one pipeline, one tuning procedure, three seeds, and four protocols: trial-level split, leave-one-subject-out, pooled two-laboratory training with matched held-out subjects, and cross-laboratory transfer. At a fixed configuration, trial-level splits overstated the subject-held-out coefficient of determination by +0.040 to +0.194; on occupational data the overstatement exceeded every difference between architectures. With subjects held out, the three models reached values between 0.583 and 0.804; the leader differed between datasets, and gaps lay within subject-to-subject variability. Pooling the laboratories changed neither dataset on matched subjects. Transfer between laboratories was below chance in both directions, including the vertical force, which no in-plane rotation can affect. Leaving force targets in the plate frame while inputs were heading-aligned cost 0.57 of locomotion anteroposterior-force explained variance, enough to make that force look hardest; a Newton's-law correlation detected the mismatch before training. Split protocol and target frame changed reported accuracy more than architecture did. Learned force estimators should be validated with subjects held out, checked for frame consistency before training, and tested across laboratories before deployment claims are made.
Anti-cancer drug response prediction (DRP) is critically important for precision oncology and therapeutic development. Currently, most DRP models are developed using drug screening data from immortalized cancer cell lines, which can differ substantially from patient tumors at the genomic and molecular levels, even though patient tumor response is the ultimate prediction target. To help bridge this gap and bring DRP models closer to their intended clinical application, we conducted four drug screening studies using patient-derived organoids (PDOs), which more faithfully recapitulate patient tumors than immortalized cell lines. These studies generated data for improving and evaluating DRP models, including multi-omics profiles of PDO lines, drug feature data, and treatment response measurements. Using these data, we retrained and evaluated multiple publicly available DRP models under three model-building scenarios: training models using cell line data only, training models using combined cell line and PDO data, and pretraining models on cell line data followed by fine-tuning on PDO data. We then evaluated and compared model prediction performance on PDOs across these analysis scenarios. In addition, we also assessed the consistency of drug screening viability data generated by different laboratories using different assay technologies and/or experimental protocols.
Whether an action-conditioned world model responds to actions is usually checked by replacing or removing the actions and measuring the loss in accuracy. Taking biological experiments as the guiding case, we show that such diagnostics report action effects where there are none: under autocorrelated designs, future actions stand in for history the model does not see. When the design law is known, a conditional randomization test written as an e-value is valid in finite samples whatever the model. A second failure arises whenever many sites share one action sequence, as in world models: the pooled score turns small model errors along the action contrast into an e-value near zero, so a valid test loses its power although the effect is real. Tempering the score changes the bet, not the validity. The log e-value is concave in the tempering factor and zero at zero, so too small a factor costs little and too large a one can cost everything; every single factor we tried, estimated or tuned, failed in some setting, and mixtures over a fixed grid never did. Our own preregistered analysis of optogenetic ERK experiments failed by pooling; a tempered statistic, registered before the data were obtained, then rejected the null on a never-opened field (E = 1.1e10). In three registered tests on models trained by others, the pooled test rejected in 0 of 96 units of a diffusion model, and reached the power bar in 10 of 26 Atari games under IRIS's token likelihood and 1 of 6 control tasks under TD-MPC2; mixtures fixed in advance reached it in 96 units, 24 games and 6 tasks. On recorded robot video with iVideoGPT the mixture rejected in 155 of 159 trajectories; the pooled test did not collapse there (143). Under exact nulls the statistics made 14 rejections in 26,664 tests.
DNA methylation (DNAm) serves as one of the most robust molecular biomarkers of biological aging. While conventional epigenetic clocks accurately predict chronological age from high-dimensional CpG profiles, they treat aging as a static regression task, meaning they can only output a single score rather than simulating how an entire profile continuously changes over time. To reconstruct these continuous dynamics, we frame lifelong human epigenetic aging as a trajectory inference problem across discrete age snapshots derived from widely available cross-sectional data. We introduce a two-stage computational pipeline: first, an age-regularized Variational Autoencoder (VAE) maps high-dimensional CpG profiles onto a chronologically ordered latent manifold while preserving a generative decoder bridge back to the original methylation space. Second, we model the continuous movement across this latent space via Regularized Unbalanced Optimal Transport (RUOT) that unifies deterministic drift, random diffusion, and non-conservative mass changes. By resolving this RUOT formulation using the DeepRUOT framework, our model fluidly accommodates population-level density shifts like survivorship bias and cellular attrition without requiring rigid biological priors. Evaluated on a large-scale, 80-year pan-tissue dataset, our model demonstrates robust distribution interpolation and uncovers a prominent late-life surge in the learned growth field that mathematically captures the variance expansion driven by stochastic epigenetic drift. Finally, by decoding continuous latent paths back to individual CpG sites, we reconstruct and empirically verify distinct biological aging archetypes, offering a rigorous, generative paradigm for simulating human molecular aging.
Designing functional biological sequences requires navigating vast discrete spaces under strict evolutionary and biophysical constraints. Discrete Flow Matching (DFM) offers a generative framework over such spaces, but existing approaches rely on biologically uninformative couplings and offer limited flexibility for variable-length sequence generation and fine-grained control. We propose a structured coupling that encodes domain-specific preferences among sequence elements, biasing the source distribution toward plausible regions without modifying the flow objective or training procedure. Building on this, we introduce a latent edit-based rate parameterization that models variable-length generation via edit operations conditioned on a shared global latent, akin to a latent variable model, while remaining tractable. We further introduce a latent classifier-free guidance mechanism that steers generation coherently in continuous latent space, along with Dirichlet-prior temperature scaling for test-time control over edit operations. Our method achieves state-of-the-art performance across diverse biological sequence tasks, including density estimation, unconditional and conditional DNA sequence generation, and peptide sequence generation. Our code is available at https://github.com/Aalto-QuML/FlexFlow.
Spatial transcriptomics (ST) enables spatially resolved gene profiling but remains expensive and low-throughput, limiting large-cohort studies and routine clinical use. Predicting spatial gene expression from routine hematoxylin and eosin (H&E) slides is a promising alternative, yet under realistic leave-one-slide-out evaluation, existing models often suffer from slide-level appearance shifts and regression-driven over-smoothing that suppress biologically meaningful variation. CHRep is a two-phase framework for robust histology-to-expression prediction. In the training phase, CHRep learns a structure-aware representation by jointly optimizing correlation-aware regression, symmetric image-expression alignment, and coordinate-induced spatial topology regularization. In the inference phase, cross-slide robustness is improved without backbone fine-tuning through a lightweight calibration module trained on the training slides, which combines a non-parametric estimate from a training gallery with a magnitude-regularized correction module. Unlike prior embedding-alignment or retrieval-based transfer methods that rely on a single prediction route, CHRep couples topology-preserving representation learning with post-hoc calibration, enabling stable neighborhood retrieval and controlled bias correction under slide-level shifts. Across the three cohorts, CHRep consistently improves gene-wise correlation under leave-one-slide-out evaluation, with the largest gains observed on Alex+10x. Relative to HAGE, the Pearson correlation coefficient on all considered genes [PCC(ACG)] increases by 4.0% on cSCC and 9.8% on HER2+. Relative to mclSTExp, PCC(ACG) further improves by 39.5% on Alex+10x, together with 9.7% and 9.0% reductions in mean squared error (MSE) and mean absolute error (MAE), respectively.
We study semisupervised mean estimation with a small labeled sample, a large unlabeled sample, and a black-box prediction model whose output may be miscalibrated. A standard approach in this setting is augmented inverse-probability weighting (AIPW) [Robins et al., 1994], which protects against prediction-model misspecification but may be inefficient when the prediction score is poorly aligned with the outcome scale. We introduce Calibrated Prediction-Powered Inference, which post-hoc calibrates the prediction score on the labeled sample before using it for semisupervised estimation. This simple step requires no retraining and can calibeat the original score, improving it both as a predictor of the outcome and as a regression adjustment for semisupervised inference. We study both linear and isotonic calibration. For isotonic calibration, we establish first-order optimality guarantees: isotonic post-processing can improve predictive accuracy and estimator efficiency relative to the original score and simpler post-processing rules, while replacing the fitted isotonic score by its conditional-mean oracle yields no additional first-order variance reduction. For linear calibration, we show first-order equivalence to PPI++. We also clarify the relationship between existing estimators, showing that the original PPI estimator is a special case of AIPW and can be inefficient when the prediction model is accurate, while PPI++ is AIPW with empirical efficiency maximization [Rubin and van der Laan, 2008]. In simulations and real-data experiments, our calibrated estimators often outperform PPI and are competitive with, or outperform, AIPW and PPI++. We provide an accompanying Python package, ppi_aipw, at https://larsvanderlaan.github.io/ppi-aipw/.
Protein language models (pLMs) produce highly accurate contact maps from single-sequence inputs. People often take this accuracy as proof that these models implicitly learn invariant coevolutionary statistics, extracting the same signals as classical alignment-based methods like Potts. In this work, we introduce a diagnostic framework around a global bijective relabeling of the amino-acid alphabet. This transformation permutes residue letters while leaving all alignment-level information, column entropies, and pairwise edit distances unchanged. Pseudolikelihood Potts models and inverse-covariance estimators remain invariant under this transformation, but pLMs fail the test. Across multiple architectures (ESM-2, MSA Transformer, AMPLIFY) and model scales (8M to 3B parameters), pLM contact maps collapse under arbitrary relabeling. Using Hamming distance controls and biochemical grouping interventions, we attempt to isolate what likely causes this degradation and find that these models rely on explicit, canonical residue identities (``alphabet anchoring'') rather than pure coevolutionary statistics. Increasing scale makes models more responsive to biochemical classes, but their performance still degrades under relabeling. Our findings show a gap between how pLMs and classical coevolutionary models compute contacts. More broadly, our work shows how diagnostic perturbations that leave classical models provably unchanged can expose hidden representation biases and prevent over-reliance on neural black boxes in biological modeling.
Predicting how a ligand binds to its target is central to small-molecule drug discovery. Modern cofolding models can generate diverse candidate poses, but reliably ranking these candidates remains challenging. We introduce Pyrite, a pairwise pose-ranking benchmark comprising 1,500 pose pairs across diverse protein-ligand complexes. Expert chemists equipped with 3D visualization tools and physics-based scores perform near chance, highlighting the difficulty of interpreting available chemical signals. To investigate automated ranking, we introduce PyriteRep, a structured representation of molecular geometry, protein-ligand interactions, and physicochemical scores, and develop two LLM-based agents: PyriteReasoner, which directly compares poses through text-based reasoning, and PyriteFeaturizer, which generates executable code to construct features for logistic regression, optionally incorporating features derived from LLM subcalls. These agents achieve up to 67% and 74% accuracy, respectively, exceeding observed expert performance. LLM-guided feature discovery generally outperforms direct reasoning, while Group Relative Policy Optimization (GRPO) improves open-weight models, enabling them to match or surpass closed-source frontier models in our evaluations. These results establish Pyrite as a challenging test of pose-ranking ability and highlight LLM-guided feature discovery as a promising approach to interpret and combine the outputs of computational chemistry tools.
Generative models are increasingly used to propose molecules for drug discovery. They can produce far more candidates than can be experimentally tested, making computational selection essential. Most selection methods rely on predicted molecular properties, such as potency and toxicity, but overlook how these properties translate into drug exposure at a given dose. As a result, molecules that appear promising on paper may require impractical doses or raise safety concerns. We investigate this gap by using physiologically based pharmacokinetic (PBPK) simulations to re-evaluate molecules selected from three PARP1-targeted generative pipelines. Of 60 shortlisted molecules, seven fail to achieve sufficient target exposure, while 28% of the remaining candidates show predicted safety risks at their required dose. Moreover, 85% of candidates with an achievable dose are outperformed by molecules overlooked during static selection. These findings suggest that accounting for dose and exposure can substantially change which generated molecules are considered promising for drug development.
Accurate cancer survival prediction remains challenging due to tumor heterogeneity. Although multimodal survival models integrate clinical and molecular data, they often overlook evolutionary information encoded in genomic alterations. To address this gap, we developed eTime, a 38-dimensional evolutionary representation comprising eight feature groups that characterize clonal and subclonal mutation burden, mutational spectra, functional consequences, copy-number alterations, and anoikis-related genomic changes. We further developed EvoSurv, a multimodal survival framework integrating eTime with clinical variables and RNA expression. Across six TCGA cancer cohorts comprising 2,782 patients, EvoSurv achieved the highest mean concordance index (C-index) in all six cancers, averaging 0.663 and outperforming three state-of-the-art survival prediction methods. More importantly, we assessed whether evolutionary features provide additional prognostic information beyond clinical and transcriptomic predictors. eTime remained significantly associated with survival in head and neck squamous cell carcinoma (HNSC) and liver hepatocellular carcinoma (LIHC) after adjustment for clinical variables and RNA-derived risk scores. Feature-group analyses further revealed cancer-specific prognostic patterns. In HNSC, T>G substitutions were associated with increased mortality risk after clinical and molecular adjustment, including in HPV-negative patients, with supporting evidence from an independent CPTAC-HNSCC cohort. Overall, these findings demonstrate that eTime captures survival-associated evolutionary information and highlight its potential to provide prognostic value beyond clinical and transcriptomic predictors.
Biological systems can form complex three-dimensional structures through the collective behavior of agents that share a common update rule and operate without central control. How such distributed control gives rise to precise global patterns remains a central question not only in developmental biology but also in distributed robotics, programmable matter, and multi-agent learning. Here, we introduce DiffeoMorph, an end-to-end differentiable framework for learning a morphogenesis protocol that guides a population of agents to morph into a target 3D shape. Each agent updates its position and internal state using an SE(3)-equivariant graph neural network, based on its own internal state and signals received from other agents. To train this system, we introduce a new shape-matching loss based on 3D Zernike polynomials, which compares the predicted and target shapes as continuous spatial distributions, not as discrete point clouds, and is invariant to agent ordering, number of agents, and global orientation. To achieve rotation invariance while preserving reflection sensitivity, we include an alignment step that optimally rotates the predicted Zernike spectrum to match the target before computing the loss. We perform benchmarking to establish the advantages of our shape-matching loss over other standard distance metrics for shape comparison tasks. We then demonstrate that DiffeoMorph can form a range of complex shapes from minimally patterned initial conditions. DiffeoMorph provides a general framework for learning distributed control strategies for morphogenesis, swarm robotics, and programmable self-assembly.
Mature, germinal centre-bearing tertiary lymphoid structures (TLS) and mature lymph node follicles (secondary lymphoid structures, SLS) sustain the plasma cell response that shapes outcome and immunotherapy benefit in lung cancer. How this loco-regional axis differs between neoadjuvant-treated and therapy-naive tumors is poorly defined. We analyzed H&E whole-slide images of resected tumors and paired proximal and distal lymph nodes from matched neoadjuvant-treated (neoLUSC, n=20) and therapy-naive (naivLUSC, n=20) lung squamous cell carcinoma (LUSC) patients. Aignostics Atlas H&E Tissue Analysis (HETA) models quantified cell densities per tissue compartment and cell-cell co-localization; a dedicated deep-learning model validated against pathologist annotations detected TLS and SLS and graded their maturity. Prognostic value was tested in an independent cohort (n=458; 46 treated). In neoLUSC tumors, stromal plasma cell density was 72% (p=0.004) and stromal lymphocyte density 48% (p=0.015) lower than in naivLUSC; macrophages and granulocytes did not differ significantly. Total and immature TLS did not differ, whereas mature TLS in the tumor (p=0.034) and mature SLS in the tumor-distal lymph node (p=0.003) were lower in neoLUSC patients. Peri-TLS plasma cell density correlated with tumor lymphocyte density in naivLUSC (rho=0.54, p=0.017) but not in neoLUSC tumors (rho=0.07). In the validation cohort, stromal plasma cell density (HR 0.69, p=0.011) and lymphocyte-plasma cell co-localization (HR 0.66, p=0.004) were associated with longer survival, and neoadjuvant status with shorter survival (HR 1.50, p=0.026). Neoadjuvant therapy is thus associated with fewer mature lymphoid structures and weaker coupling of the humoral compartment to the tumor rather than uniformly lower immune readouts. The stromal plasma cell/co-localization axis is a candidate prognostic biomarker.
Current single-cell clustering pipelines discard most genes and can yield inconsistent clusters across random seeds. We introduce KQE-2, a training-free hybrid quantum-classical framework for reproducible whole-transcriptome clustering. KQE-2 maps cells into entanglement-free quantum states, enabling exact feature evaluation on classical hardware and direct checks on a quantum processor. Theoretically, we prove that removing entanglement is necessary and sufficient to bound single-qubit feature variance locally. Practically, while retraining scVI shifted 14% of cells and altered rare-type markers and drug targets, KQE-2 delivered identical biology across runs. On standard highly variable genes, the quantum encoding alone raised accuracy on a developing cortex from 0.61 to 0.79. KQE-2 scales to 11.4 million nuclei on one server, outperforming the compared methods on eight of ten complete atlases (and within the best method's run-to-run range on two), and leads on six even under standard downstream clustering.
The rapid growth of scientific literature and the high heterogeneity of biological terminology and mechanistic descriptions make it difficult for researchers to integrate scattered research evidence, identify connections among biological events, and construct mechanistic networks. Here, we developed a high-throughput human-AI hybrid framework, AOP-Smart-Finder, for biological event discovery, relationship mining, and Adverse Outcome Pathway (AOP) construction and evidence mapping from large-scale literature. This framework integrates Large Language Models (LLMs), AOP-Smart retrieval-augmented generation (RAG) technology, and Python-based automation, supporting two analysis modes: one based on a single compound and the other based on a predefined set of biological events. In the di(2-ethylhexyl) phthalate (DEHP) case, the system processed 12,983 articles within 30 min and included 1,093 of them, from which it extracted 1,589 biological events (including 458 official key events [KEs] and 1,131 candidate KEs) and 2,519 key event relationships (KERs). In the amyotrophic lateral sclerosis (ALS) case, the system identified 55 stressors from 437 articles and extracted 278 biological events and 548 event relationships. Following manual curation and Weight of Evidence (WoE) evaluation, a mechanistic network comprising 16 KEs and 23 KERs was ultimately constructed. This framework can either use existing AOPs as knowledge guidance for mechanistic expansion and evidence supplementation, or discover novel event connections and construct mechanistic networks around user-specified research topics. It provides a high-throughput, traceable, human-AI hybrid methodology for complex biological mechanism elucidation and AOP discovery driven by large-scale literature.
Public scientific datasets rarely arrive analysis-ready. They arrive as messy folders of wide matrices, long tables, spreadsheets, and per-cohort files whose keys and encodings differ, and every reanalysis must reconnect them first. A coding agent can automate this, but its mistakes can look plausible, and without an audit trail, a mistake that corrupts data can go uncaught. We present an AI-assisted ingestion pipeline built on one rule: LLM agents propose, deterministic code verifies, and humans decide. Narrow agents see compact, citable evidence about the files, never the data, and return typed proposals. A verifier with 39 check types tests each proposal against that evidence in two loops, one for the schema's structure and one for its meaning, and a human reviewer approves the schema and the load. Code compiles PostgreSQL DDL, reshapes the data, quarantines rows it cannot place, records provenance, and reconciles every staged row against the database. On four public biomedical datasets, one held out from development, 9 of 20 first proposals failed a check and every failure was caught: the loops repaired eight, and a one-line reviewer edit fixed the ninth. All 45.1 million values loaded with zero reconciliation gap. Discovering a dataset's structure took 4-7 LLM calls and preparing its data at most one more, costing under $0.50 per dataset. In a separate test, each of nine planted data errors was quarantined with a reason or recorded in an audit log.
Modern observational biomedical datasets contain increasingly extensive baseline and outcome measurements, yet detecting a clinically meaningful treatment effect remains difficult because investigators must identify an outcome that captures treatment response while adjusting for confounding distributed across numerous noisy baseline variables. We introduce SCOUT (Score-based Causal Outcome Learning Using Treatment signals), a framework that learns a clinically interpretable, treatment-responsive outcome while adjusting for confounding throughout the learning process. SCOUT constructs low-dimensional adjustment representations of high-dimensional baseline measurements to estimate the average treatment effect (ATE) and conditional average treatment effect (CATE) for the learned outcome. Importantly, the final adjustment space is at most two-dimensional, allowing treatment-effect heterogeneity to be estimated in this space rather than in the full high-dimensional covariate space. SCOUT also introduces a fourth-order statistic for testing treatment-effect heterogeneity that emphasizes unusually large deviations from the ATE. In synthetic and semi-synthetic experiments, the algorithm achieved competitive ATE estimation accuracy and substantially lower CATE estimation error. In an observational study of patients with bipolar disorder, SCOUT identified a clinically recognizable lithium-associated adverse-effect profile, detecting both a significant ATE (Holm-adjusted $p$ = 0.0026) and significant treatment-effect heterogeneity (Holm-adjusted $p$ = 0.033).These results demonstrate how jointly learning outcomes and controlling for confounding can facilitate the discovery of treatment effects in high-dimensional observational data.
Cell types are organized by taxonomic relationships that define lineages in the so-called cell ontology. However, most existing foundation models typically ignore cell-type lineages encoded in this cell ontology. We introduce a biologically informed foundation model called scOntoFM, which embeds hierarchical cell ontology into representation learning. Using Lowest Common Ancestor (LCA) distances from the ontology graph, our framework pairs efficient offline triplet sampling with hierarchical cell-ontology learning. This approach jointly preserves local neighborhoods and shapes the global embedding geometry to reflect the ontology structure. Across diverse cell- and gene-level benchmarks, the model consistently improves performance, with especially strong gains in zero-shot settings. Our model also balances batch integration with biological structure preservation, providing complementary perspectives to existing embedding evaluation frameworks and supporting scalable biological discovery.
Accurate prediction of enzyme kinetic parameters can support enzyme engineering and metabolic modeling by complementing experimental characterization. Existing prediction methods often pool residue-level protein features before combining them with substrate features. Such substrate-independent pooling cannot adapt residue weighting to the substrate during aggregation, potentially diluting information relevant to a specific enzyme-substrate pair. Since the contributions of individual residues to catalysis can depend on the substrate, substrate-conditioned aggregation offers a way to emphasize residue features relevant to the prediction. We introduce KineCouple, which uses substrate queries and residue-level keys and values in cross-attention, allowing the substrate to guide residue selection before aggregation. The model preserves the global protein representation and adds a gated correction defined as the difference between the conditioned readout and a uniform readout. ProtT5 and MolT5 remain frozen, and separate models predict kcat and Km on a logarithmic scale. In pooled out-of-fold predictions on the CataPro benchmarks, KineCouple reduces kcat RMSE from 1.333 to 1.330 and increases PCC from 0.492 to 0.496 relative to reproduced CataPro. For Km, RMSE decreases from 0.998 to 0.997 and PCC increases from 0.633 to 0.634. Spearman correlation also improves for both tasks.
Understanding how cells respond to perturbations over time is central to cell biology and drug discovery. Live-cell imaging captures the dynamics of perturbation-induced phenotypes in individual cells. While fluorescence imaging can visualize specific molecular phenotypes, labeling and repeated illumination often affect cell health. Virtual staining offers an alternative by predicting these fluorescence images from images acquired without fluorescent labels. Although many virtual staining models have been developed, their performance on volumetric time-lapse data remains poorly characterized. We address this gap with an evaluation suite for 3D time-lapse virtual staining of live cells. The suite combines paired 3D time-lapse images acquired with and without fluorescent labels, a set of baseline models, and three metric tiers measuring pixel-level fidelity, organelle segmentation performance, and single-cell phenotype similarity. Using this evaluation suite, we study metric and model behavior across 3 cell types, 2 microscopes, 4 organelles, and 3 perturbation states. We find that regression baselines better predict the spatial localization of target organelles, whereas the generative baseline better captures the distribution of phenotypes across cells. These differences expose trade-offs between structural and phenotypic fidelity that are obscured by single-metric evaluation and suggest how virtual staining models can be developed for diverse downstream tasks such as localization, segmentation, tracking, and phenotypic profiling. We release the curated datasets, baseline checkpoints, and evaluation code as part of DynaCell, a broader framework for image-based profiling of cell-state dynamics.