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Fu, D.

Publications and source records attributed to Fu, D..

At least 19 recordsLinked to original sources

Evaluating agentic AI for biological discovery in autonomous and copilot settings

Advances in large language models (LLMs)-based artificial intelligence (AI) agents have improved their ability to execute structured analytical workflows, including standard bioinformatic pipelines for biological discovery. However, computational biology rarely consists of deterministic pipeline execution alone. Biological datasets are heterogeneous and noisy, and meaningful discovery often requires open-ended hypothesis generation and iterative reasoning over multimodal evidence. These challenges are particularly evident in multi-omic studies, where paired molecular modalities and heterogeneous clinical contexts create both opportunities and obstacles for discovery. The extent to which emerging agentic AI systems can support or automate this mode of scientific discovery remains poorly understood. Here, we systematically evaluated the capabilities and limitations of agentic AI for biological discovery using multi-omic single cell datasets spanning 11 cancer types. We developed the Multistep Multimodal Multiomic Agentic (M3A) Framework to support LLM-driven reasoning over persistent multimodal data states and to capture agentic reasoning behavior in autonomous and human-AI copilot settings. Using this framework, we assessed AI agents across complementary tasks, including autonomous cell-type annotation, generation of falsifiable biological hypotheses from gene programs, and copilot experiments testing the effect of human involvement and domain expertise. We found that current AI agents are effective at broad, systemic exploration of complex data, whereas domain experts remain critical for methodological guidance and biological synthesis across analyses. Together, our results delineate the current potential and boundaries of agentic AI in computational biology, and establish a framework for evaluating AI systems designed to support biological discovery.

cancer biology↗

Degradation of net ecosystem carbon balance in cool-temperate forests by sika deer-induced stand structure alterations and subsequent soil erosion

Although natural forests sequester carbon, this function may decline under chronic herbivory by abundant ungulates (hereafter overbrowsing). Specifically, overbrowsing alters stand structure, potentially impair carbon exchanges related to the vegetation. Further, overbrowsing may also accelerate soil erosion, especially in heavy-rainfall regions like Monsoon Asia. We quantified these impacts by estimating net ecosystem carbon balance (NECB; g C m-2 yr-1) by subtracting heterotrophic respiration (Rh) and lateral carbon export via erosion (Se) from net primary production (Pn) in southern Kyushu, Japan. Here, about 40-years of overbrowsing by sika deer (Cervus nippon) altered mixed broadleaf-conifer stands with presence of understory (PU) into stands with no understory (NU), then further altered into stands dominated by unpalatable shrublands (SR) or stands with canopy gaps (CG). The PU maintained a positive NECB (plot mean = 307.0 g C m-2 yr-1) because high Pn (721.9) exceeded the sum of Rh (175.4) and Se (239.5). Alteration from PU into NU converted NECB to negative (-98.2 g C m-2 yr-1). This was because the suppressed Pn (400.2 g C m-2 yr-1) could not offset the sum of Rh (170.6) and Se (327.7). Further degradation into CG caused a profound negative NECB (-894.4 g C m-2 yr-1), where Pn (71.9) offset only 7% of the sum of surging Rh (464.8) and Se (501.5). Alteration into SR showed a partially recovered NECB (97.3 g C m-2 yr-1), driven by shrub growth (Pn; 554.5, Rh; 175.4, Se; 239.5). However, this recovery is still limited given that lowered shrub biomass and prior topsoil loss via erosion. Our results validate previous findings that stand alteration from PU to SR or CG through NU leads to up to a 49% loss of ecosystem carbon stocks. Preventing stand alteration and soil erosion are key countermeasures against chronic overbrowsing and subsequent erosion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/719128v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@167aef0org.highwire.dtl.DTLVardef@e45a6org.highwire.dtl.DTLVardef@fec89borg.highwire.dtl.DTLVardef@1246bea_HPS_FORMAT_FIGEXP M_FIG C_FIG We reported that over 40 years of sika deer overbrowsing and subsequent soil erosion severely degraded the net ecosystem carbon balance (NECB) of mountain forests in Japan. The loss of understory vegetation drove the transition of intact stands into degraded states (no-understory, shrub-dominated, or canopy gaps). Based on field measurements, we quantified that this structural alteration suppressed net primary production (Pn) while increased both heterotrophic respiration (Rh) and lateral carbon loss via soil erosion (Se). Consequently, the forest shifted from a net carbon sink (+307 g C m-{superscript 2} yr-{superscript 1}) to a source (up to -894 g C m-{superscript 2} yr-{superscript 1}). These findings provide compelling empirical evidence that increasing ungulate populations, compounded by the rising frequency of heavy rainfall, may severely undermine the carbon sequestration functions traditionally expected of natural forests.

ecology↗

Coding Agents as a Mechanism for Formalizing and Transferring Domain Knowledge in DNA Origami Design

This work investigates the use of a coding agent to design and manipulate DNA origami structures through the caDNAno2 design interface. The design methodology produced by the agent is saved as transferable, repeatable text-file instructions interpretable by another coding agent, converting informal domain expertise into formalized, distributable protocols. We demonstrate this through a parametric pipeline for simple single and multi-layer designs, identifying several failure modes that each required explicit domain knowledge from the human designer. Once encoded, this knowledge enabled the agent to autonomously extend designs to arbitrary lattice dimensions, generate structures to physical specifications, execute targeted edits from natural language prompts, and run the full pipeline from design through molecular dynamics simulation.

bioengineering↗

A chromatin fiber model explains cell-free DNA fragmentation signatures of active regulatory elements

Circulating cell-free DNA (cfDNA) assays are being widely adopted in oncology and maternal-fetal medicine. Patterns of cfDNA fragmentation can provide useful information about gene regulation and expression in human disease from a blood draw. Here, we demonstrate that enhancer RNA expression - a marker of enhancer activity - can be inferred from local patterns of cfDNA fragmentation. We define a transcriptional activation score (TAS) that predicts expression of enhancers and genes based on cfDNA fragment sizes and positions near transcriptional start sites (TSSs). The TAS identifies activity of cancer-associated enhancers in patients with cancer, distinguishes clinically relevant cancer subtypes, and identifies activation of enhancers associated with treatment resistance and therapy response. We propose a simple model to account for our findings based on chromatin fiber structure and the depletion of H1 histone proteins near active TSSs. Our model provides a unified framework that reconciles seemingly conflicting observations from prior fragmentomics studies. Broadly, this work enables blood-based assessments of gene regulation in cancer and non-oncologic diseases to inform pathobiology, diagnosis, and treatment selection.

genomics↗

AAV Kills Dividing Cells by Depleting PARP1 and Other DNA Damage Response Proteins

Recombinant adeno-associated virus (rAAV) is a replication-defective viral vector used in hundreds of human gene therapy trials, resulting in five FDA-approved therapies. Despite this success, rAAV-based gene therapies suffer from dose-limiting toxicities, resulting in several severe adverse reactions, including death. Previously, we discovered that rAAV rapidly kills mouse NPCs in vitro and in vivo. This vector contains a minimal genome comprised of 145-base pair inverted terminal repeats (ITRs) with a T-shaped hairpin structure that appears to be necessary and sufficient for this toxicity. However, the mechanism for AAV ITR toxicity is not known, and there have been few attempts to engineer ITRs to attenuate rAAV toxicity. In the current study, we explore the molecular mechanisms that drive dose-dependent rAAV toxicity in dividing human NPCs (hNPCs) and test whether disrupting these mechanisms mitigates this toxicity. Recombinant AAV infection induces aberrant cell cycle progression with activation of the ATM /CHK1/CHK2 pathway and expression of the DNA damage markers {gamma}H2AX and 53BP1. Affinity-based proteomics indicate that AAV ITRs bind to Poly-(ADP-Ribose)polymerase 1 (PARP1) and other DNA damage response (DDR) proteins involved in single-strand break repair (SSBR). Recombinant AAV infection attenuates poly-(ADP-ribose) (PAR) formation and mimics the antiproliferative effects of pharmacological PARP inhibitors used in cancer therapy. Moreover, treatment of hNPCs with PARP inhibitors is sufficient to reproduce many features of rAAV-induced toxicity. Finally, we demonstrate that eliminating the T-shaped hairpin within the AAV ITR reduces binding to SSBR proteins and the resulting rAAV toxicity. These findings suggest that rAAV infection induces replication stress and cell death in dividing hNPCs by functionally depleting PARP1 and other DDR proteins that are essential for DNA replication. This work fills substantial gaps in the understanding of the mechanisms of rAAV toxicity and has important implications for the development of safer rAAV-based human gene therapies. One Sentence SummaryThe rAAV genome binds to and depletes PARP1 and other SSBR proteins that are essential for DNA replication, resulting in DNA double stranded breaks, checkpoint activation, and cell death in dividing cells.

cell biology↗

Mycoelectronics: Bioprinted Living Fungal Bioelectronics for Artificial Sensation

The intelligence of the human biological system is enabled by the highly distributed sensing receptors on soft skin that can distinguish various stimulations or environmental cues, thus establishing the fundamental logic of sensing and physiological regulation or response. To replicate biological perception, two approaches have emerged: artificial nervous systems that utilize soft electronics as biomimetic receptors to convert external stimuli into frequency-encoded signals, and biohybrid solutions that integrate living cells, plants, or even live animals with electronic components to decode environmental cues for life-like sensations. However, most current biohybrid approaches for artificial sensation are based on eukaryotic cells, which suffer from slow growth, stringent culture conditions, environmental susceptibility, and short lifespans, thus limiting their integration into practical wearables or robotic sensory skins. Here, we introduce fungi-based printable "Mycoelectronics", which are created by additive bioprinting of living fungal mycelium networks onto stretchable electronics, as a practical living thermo-responsive sensory platform. This Mycoelectronics approach leverages fungis capacity for rapid biological responsiveness, cultivability with exponential growth, stability and self-healing in ambient conditions, bioprintability for scalable manufacturing, and mechanical flexibility for seamless integration with soft electronics. Critically, we discovered that the thermal responsiveness of the fungal network arises from intrinsic cellular processes--specifically, heat-induced vacuole remodeling and fusion, which modulate ionic transport and thus the electrical conductivity of the mycelial cells and networks, enabling a rapid temperature response. By bridging the gap between cell biology and soft electronics, the Mycoelectronics device with a living mycelium network functions as a thermal sensation system with rapid response and intrinsic self-healing properties, autonomously restoring sensing capabilities after damage or autonomously establishing sensor pathways in hard-to-reach locations. Furthermore, by integrating fungal thermal sensing with electronic circuits, we established a hybrid bioelectronic reflex arc that can actuate muscles and initiate diverse actions, suggesting promising applications in future neurorobotics and neuroprosthetics.

bioengineering↗

From ambiguous object to illusory faces: EEG decoding reveals a dynamic cascade of face pareidolia

Face pareidolia, perceiving faces in inanimate objects, provides a unique window into how the brain constructs perceptual meaning from ambiguous visual input. In this work, we investigated the neural dynamics of face pareidolia, testing the hypothesis that it emerges from a temporal cascade that integrates global (low spatial frequency, LSF) and local (high spatial frequency, HSF) visual information. With a pareidolia detection task, we combined event-related potentials (ERP), multivariate pattern analysis (MVPA), and representational similarity analysis (RSA) to map the evolving neural code for illusory face perception. Behaviorally, participants experienced pareidolia from both LSF and HSF stimuli, but the percept was strongest for broadband images containing both frequency bands. Electrophysiological results revealed a clear temporal progression. Early processing (<150 ms) was driven by low-level attributes, with distinct P100/N100 components and neural representations for LSF and HSF signals. A critical shift occurred around 145 ms, after which MVPA decoding could reliably distinguish between images perceived as faces and those that were not. Concurrently, RSA showed that the geometry of neural representations shifted to reflect participants subjective face-likeness ratings, rather than the initial physical properties of the stimuli. This transition to a higher-order, subjective evaluation was marked by a later N250 component, whose amplitude indexed the cognitive load of integrating the visual cues. Together, these findings demonstrate that face pareidolia is a dynamic neural cascade, from early sensory encoding to higher-order perceptual evaluation, driven by the integration of global configuration and local features of visual stimuli. Our results illuminate how the brain transforms ambiguous input into meaningful social perception, offering insight into the temporal dynamics of visual awareness.

neuroscience↗

4-Pi Stimulated Raman Scattering for Label-free Super-resolution Chemical Imaging

AO_SCPLOWBSTRACTC_SCPLOWSuper-resolution fluorescence microscopy has transformed the study of biological structures and functions beyond the diffraction limit. Unlike fluorescence methods, label-free chemical imaging, mostly based on Raman and infrared spectroscopy, provides intrinsic molecular contrast, enabling the study of biomolecules, nanostructures, drug molecules, and metabolites that cannot be easily tagged. However, while a wide range of fluorescence-based super-resolution techniques are well-established, extending super-resolution to label-free chemical imaging has remained challenging due to low signal levels and limited resolution improvement. Super-resolution stimulated Raman scattering microscopy (SRS) is most promising due to its high sensitivity and imaging speed. Similar to fluorescence, existing SRS super-resolution approaches are mostly based on either photoswitching/saturation of molecular labels or sample expansion, which suffers from poor sensitivity due to limitations in labeling density or signal dilution, respectively. Moreover, axial resolution is typically much worse than lateral resolution, yet most super-resolution SRS techniques focused on improving lateral resolution. In this work, we combine stimulated Raman scattering (SRS) with 4Pi-interferometry to significantly improve the axial resolution by nearly 7-fold. We report on the characterization of improvements in imaging sensitivity and axial resolution using 80 nm polystyrene beads. Harnessing the improved axial resolution, we demonstrate super-resolution 4Pi-SRS imaging in resolving small lipid droplet structures in mammalian cells and lipid membranes in E. coli cells. Because 4Pi-SRS uses interferometry to improve axial resolution, it is completely orthogonal to all previous super-resolution SRS techniques, including visible excitation, photoswitching, sample expansion, and computational approaches, thus it is straightforward to combine them to achieve much higher resolution chemical imaging than currently possible.

bioengineering↗

Label-Free Longitudinal Imaging of Single Cell Drug Response with a 3D-Printed Cell Culture Platform

Image-based phenotypic screening has emerged as a powerful tool for revealing single-cell heterogeneity and dynamic phenotypic responses in preclinical drug discovery. Compared to traditional static end-point assays, live-cell longitudinal imaging captures the temporal trajectories of individual cells, including transient morphological adaptations, motility shifts, and divergent subpopulation behaviors, enabling high content features and more robust early prediction of treatment outcomes. Fluorescence-based screening, while highly specific, is constrained in live-cell contexts by broad spectral overlaps (limiting multiplexing to fewer than six channels), bulky fluorophores that may perturb small-molecule interactions, and photobleaching or phototoxicity under repeated excitation. Stimulated Raman scattering (SRS) microscopy overcomes these barriers by delivering label-free, quantitative chemical contrasts alongside morphological information. Here, we present a low-cost, 3D printed cell culture platform compatible with the stringent optical requirements of SRS microscopy. This set up enables real-time drug delivery and continuous monitoring of biochemical and morphological changes in living cells during 24-hour time-lapse imaging with minimal photodamage. We outline a processing pipeline for longitudinal SRS images to extract chemical and morphological features of single live cells. Using this system, we showcase time-lapse SRS microscopy as a tool to map heterogenous drug-induced single-cell response over time, enabling the identification of varying trajectories within complex cell populations. By parallelizing multi-well perfusion with label-free chemical imaging, our approach offers a pathway toward high-throughput pharmacodynamic assays for the acceleration of phenotypic screening and personalized medicine.

bioengineering↗

YAP signaling promotes resistance to MEK and AKT inhibition in NF1-related MPNSTs

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by loss of function of the neurofibromin protein. Malignant peripheral nerve sheath tumors (MPNSTs) are a rare and deadly sarcoma with few therapeutic options that are the leading cause of death for patients with NF1. To date, no targeted therapies have been approved for MPNST treatment, highlighting the need for an understanding of adaptive signaling mechanisms that drive resistance. We developed a preclinical model of drug resistance using a cross-over drug holiday design and evaluated patterns of response and resistance to MEK and AKT inhibitors, two pathways that are dysregulated by loss of neurofibromin. We show that the mTOR and YAP/TEAD pathways are activated by MEK inhibitor exposure, yet blockade of these pathways in resistant MPNST PDX models does not significantly reduce tumor growth, despite strong in vitro synergy between trametinib and the novel TEAD inhibitor, GNE-7883. Using spatial transcriptomics, we uncovered phenotypic inertia as a key mechanism of drug resistance in MPNST, in addition to signaling plasticity. Further, we found that resistance is mediated by sustained ERK, YAP, and MYC driven transcriptional programs. In the future, preclinical studies should focus on addressing intratumoral heterogeneity and how it evolves over time.

cancer biology↗

Sequencing-based Spatial Transcriptomics with scRNA-seq Sensitivity

The advent of spatial transcriptomics has dramatically expanded our ability to study the vast network of cell-cell interactions at the molecular level in tissue. Among current methods, sequencing-based approaches have great potential in discovering because of its unbiased capture. In the last couple of years, the spatial resolution for the capture addresses has been significantly improved from 100 m to <1 m, well below the size of a mammalian cell. However, the capture efficiency has always been a pain point, [~]one order of magnitude lower than that of single cell RNA sequencing (scRNA-seq). The low capture efficiency limits the depth and breadth of its applications in the study of complex biological systems and diseases. Here, we introduce Salus Spatial transcriptomic system (Salus-STS), which provides [~]1 m capture resolution and a capture efficiency [~]1 order of magnitude higher than other current methods. Analysis with sub-cellular resolution becomes practical for sequencing-based spatial transcriptomics.

molecular biology↗

Structural impact of 3-methylcytosine modification on the anticodon stem of a neuronally-enriched arginine tRNA

All tRNAs undergo a series of chemical modifications to fold and function correctly. In mammals, the C32 nucleotide in the anticodon loop of tRNA-Arg-CCU and UCU is methylated to form 3-methylcytosine (m3C). Deficiency of m3C in arginine tRNAs has been linked to human neurodevelopmental disorders, indicating a critical biological role for m3C modification. However, the structural repercussions of m3C modification are not well understood. Here, we examine the structural effects of m3C32 modification on the anticodon stem loop (ASL) of human tRNA-Arg-UCU-4-1, a unique tRNA with enriched expression in the central nervous system. Optical melting experiments demonstrate that m3C modification can locally disrupt nearby base pairing within the ASL while simultaneously stabilizing the ASL electrostatically, resulting in little net change thermodynamically. The isoenergetic nature of the C32 - A38 pair vs the m3C32 - A38 pair may help discriminate against structures not adopting canonical C32 - A38 pairings, as most other m3C pairings are unfavorable. Furthermore, multidimensional NMR reveals that after m3C modification there are changes in hairpin loop structure and dynamics, the structure of A37, and the neighboring A31 - U39 base pair. However, these structural changes after modification are made while maintaining the shape of the C32 - A38 pairing, which is essential for efficient tRNA function in translation. These findings suggests that m3C32 modification could alter interactions of tRNA-Arg isodecoders with one or more binding partners while simultaneously maintaining the tRNAs ability to function in translation.

biochemistry↗

Stratified analysis identifies HIF-2α as a therapeutic target for highly immune-infiltrated melanomas

While immune-checkpoint blockade (ICB) has revolutionized treatment of metastatic melanoma over the last decade, the identification of broadly applicable robust biomarkers has been challenging, driven in large part by the heterogeneity of ICB regimens and patient and tumor characteristics. To disentangle these features, we performed a standardized meta-analysis of eight cohorts of patients treated with anti-PD-1 (n=290), anti-CTLA-4 (n=175), and combination anti-PD-1/anti-CTLA-4 (n=51) with RNA sequencing of pre-treatment tumor and clinical annotations. Stratifying by immune-high vs -low tumors, we found that surprisingly, high immune infiltrate was a biomarker for response to combination ICB, but not anti-PD-1 alone. Additionally, hypoxia-related signatures were associated with non-response to anti-PD-1, but only amongst immune infiltrate-high melanomas. In a cohort of scRNA-seq of patients with metastatic melanoma, hypoxia also correlated with immunosuppression and changes in tumor-stromal communication in the tumor microenvironment (TME). Clinically actionable targets of hypoxia signaling were also uniquely expressed across different cell types. We focused on one such target, HIF-2, which was specifically upregulated in endothelial cells and fibroblasts but not in immune cells or tumor cells. HIF-2 inhibition, in combination with anti-PD-1, enhanced tumor growth control in pre-clinical models, but only in a more immune-infiltrated melanoma model. Our work demonstrates how careful stratification by clinical and molecular characteristics can be leveraged to derive meaningful biological insights and lead to the rational discovery of novel clinical targets for combination therapy.

cancer biology↗

Manipulation of the brown glume and internode 1 gene leads to alterations in lignified tissue coloration, lignification, and pathogen resistance in wheat

Lignin is a crucial component of the cell wall, providing mechanical support and protection against biotic and abiotic stresses. However, little is known about wheat lignin-related mutants and their roles in pathogen defense. In this study, we identified an ethyl methanesulfonate (EMS)-derived Aegilops tauschii mutant named brown glume and internode 1 (bgi1), which exhibits reddish-brown pigmentation in various tissues, including internodes, spikes, and glumes. Using map-based cloning and single nucleotide polymorphism (SNP) analysis, we identified AET6Gv20438400 (BGI1) as the leading candidate gene, encoding the TaCAD1 protein. The mutation occurred in the splice acceptor site of the first intron, resulting in a premature stop codon in BGI1. We validated the function of BGI1 using loss-of-function EMS and gene editing knockout mutants, both of which displayed reddish-brown pigmentation in lignified tissues. BGI1 knockout mutants exhibited reduced lignin content and shearing force relative to wild type, while BGI1 overexpression transgenic plants showed increased lignin content and enhanced disease resistance against common root rot and Fusarium crown rot. We confirmed that BGI1 exhibits CAD activity both in vitro and in vivo, playing an important role in lignin biosynthesis. BGI1 was highly expressed in the stem and spike and localized in the cytoplasm. Transcriptome analysis revealed the regulatory networks associated with BGI1. Finally, we demonstrated that BGI1 interacts with TaPYL-1D, potentially involved in the abscisic acid signaling pathway. The identification and functional characterization of BGI1 significantly advance our understanding of CAD proteins in lignin biosynthesis and plant defense against pathogen infection in wheat.

plant biology↗

Wheat stripe rust resistance gene Yr9, derived from rye, is a CC-NBS-LRR gene in a highly conserved NLR cluster

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a significant threat to global wheat production. Genetic resistance plays a crucial role in controlling this disease. Among wheat breeding innovations, the wheat-rye 1BL.1RS translocations are notable for introducing alien genetic diversity, partly due to the presence of the stripe rust resistance gene Yr9 on 1RS. To clone Yr9, we first identified four Pst-susceptible mutants from Lumai 15, which carries the 1BL.1RS translocation and Yr9. Using these mutants, we performed Sequencing Trait-Associated Mutations (STAM). A single candidate gene, YrChr1B, was identified within the Yr9 locus and later confirmed as Yr9 through genetic complementation and gene editing. The Yr9 gene encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR or NLR) protein and is part of a 14-member NLR gene cluster. This cluster is conserved among Triticeae species and is an ortholog of the barley Mla locus. Cloning Yr9 expands the genetic resources available for molecular wheat breeding aimed at durable and broad-spectrum disease resistance.

plant biology↗

BRD4 phosphorylation regulates the structure of chromatin nanodomains

The interplay between chromatin structure and phase-separating proteins is an emerging topic in cell biology with implications for understanding disease states. Here, we investigate the functional relationship between bromodomain protein 4 (BRD4) and chromatin architecture. By combining molecular dynamics simulations with live-cell imaging, we demonstrate that BRD4, when mutated at specific N-terminus sites, significantly impacts nucleosome nanodomain (NN) organization and dynamics. Our findings reveal that enhanced chromatin binding activity of BRD4 condenses NNs, while both loss or gain of BRD4 chromatin binding reduced diffusion of single nucleosomes, suggesting a role for BRD4 in the regulation of nanoscale chromatin architecture and the chromatin microenvironment. These observations shed light on the nuanced regulation of chromatin structure by BRD4, offering insights into its role in maintaining the nuclear architecture and transcriptional activity.

biophysics↗

Mature and migratory dendritic cells promote immune infiltration and response to anti-PD-1 checkpoint blockade in metastatic melanoma

Immune checkpoint inhibitors (ICIs) have heralded a remarkable shift in cancer care, significantly extending survival for advanced cancer patients. However, despite their remarkable clinical successes, a substantial majority of patients fail to achieve a lasting response to ICIs. To address this challenge and gain insights into the complex landscape of the tumor microenvironment (TME), we conducted an extensive analysis using single-cell RNA sequencing (scRNA; [~]216K cells across 39 samples) and single-nucleus transposase-accessible chromatin sequencing (snATAC; [~]43K cells from 15 samples) in a metastatic melanoma cohort. This systematic approach delineates 14 distinct cell types and 55 cell subtypes, including the identification of 15 transcriptional hallmarks of malignant cells. Through correlation analysis of cell subtype proportions, we unveiled six distinct clusters associated with varying tumor responses. Particularly intriguing was the identification of the mature DC enriched in immunoregulatory molecules (mregDC) subtype exhibiting correlations with naive T and B cells, forming an anti-tumor program that underscores the importance of multiple immune cell types in mediating anti-tumor immunity. Notably, we found that mregDC abundance represents a good prognostic predictor of progression-free survival (PFS) in the context of ICI treatment, and when combined with the TCF7+/- CD8 T cell ratio, it reliably predicts patient PFS across treatments beyond ICI. We validated our findings using an independent cohort of 274 ICI-treated melanoma samples analyzed using tissue-level expression. We next compared mregDCs and conventional dendritic cell types 1 and 2 (cDC1 and cDC2) using transcriptome signature, differentiation trajectory, interactome, cytokine milieu, and epigenome landscape analyses. This comparative analysis shed light on the unique attributes of mregDCs within the TME. Finally, we investigated cell type/subtype-specific genes, pathways, immune response enrichment, and ligand-receptor interactions closely associated with the proportions of mregDCs within the TME. These molecular and cellular insights, with their critical roles in enhancing the immune response against cancer, offer valuable prospects for predicting the efficacy of ICI regimens, and potentially guiding the selection of rational combinatorial therapies.

cancer biology↗

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. The method has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 {micro}g sample input, with 235 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

biochemistry↗