Biology subjects

He, Y.

Publications and source records attributed to He, Y..

At least 199 records · Page 11Linked to original sources

bioRxiv2025

Beyond Motility: A Multiparametric Fitness Assay Framework to Evaluate Anthelmintics in Nippostrongylus brasiliensis

We adapted a multiparametric fitness assay panel--including ATP quantification, larval growth, pigmentation, and cuticle permeability--to assess Nippostrongylus brasiliensis viability in vitro. To validate this system, we benchmarked two known anthelmintics with distinct modes of action: quinidine, a heme detoxification disruptor, and pyrantel pamoate, a neuromuscular antagonist. We then applied this framework to NCR247, a plant-derived heme-sequestering peptide with proposed antiparasitic activity. While the assay panel sensitively detected physiological disruption by known anthelmintics, NCR247 did not significantly impair parasite fitness across any tested parameter. These findings highlight the utility of integrated viability assays and suggest further in vivo or delivery-optimized studies may be necessary to evaluate NCR247 efficacy.

molecular biology↗

bioRxiv2025

Single oocyte full-length isoform sequencing unveils the impact of transposable elements on RNA diversity and stability during oocyte maturation

The oocyte-specific isoforms play crucial roles in oocyte maturation, while current understanding of the oocyte transcriptome is mainly focused on gene level. Here, we utilized single cell full-length isoform sequencing based on third generation sequencing to detect entire transcripts in human and mouse oocytes. Isoform diversity during oocyte maturation was systematically profiled, including 6,736 and 4,902 putative novel human and mouse transcripts, respectively. More than half of novel isoforms were categorized as the novel-not-in-catalog (NNC) and may serve specific functions in oocytes, including novel isoforms of ARHGAP18, colocalized with microtubules and targeted knockdown disrupting oocyte maturation. Moreover, over 25% of NNC isoforms were derived from transposable elements (TEs), and their incorporation within transcripts could enhance isoform stability during oocyte maturation. Altogether, our findings represent a valuable resource showcasing the complexity and diversity of RNA isoforms in oocytes, as well as TE co-option for novel isoform generation and isoform stability enhancement.

developmental biology↗

bioRxiv2025

Phosphorylation-inducing chimera rewires oncogenic kinase to trigger apoptosis

The active sites electric field is integral to enzymatic catalysis (e.g., substrate recognition) and nature employs charge-altering post-translational modifications (e.g., phosphorylation) to perturb this electric field and regulate enzymes. A chromosomal translocation converts Abelson kinase (ABL) to BCR-ABL, whose hyperactivity drives several cancers. Here, we developed a small molecule, BRD8833, that induces BCR-ABL phosphorylation, which perturbs its active sites electric field with loss of hyperactivity. Unlike "occupancy-driven" inhibitors that require stoichiometric concentrations, BRD8833 operates through an event-driven, substoichiometric mechanism by inducing proximity between two BCR-ABL molecules to trigger the inhibitory phosphorylation and selective apoptosis of BCR-ABL-dependent cancer cells. Furthermore, BRD8833 is effective against other oncogenic ABL fusions or clinically observed resistance mutations, including those to occupancy-driven drugs with the same binding site as BRD8833, suggesting differences in their resistance mechanisms. These studies lay the foundation for electric-field and "event-driven" modalities to control hyperactive enzymes with orthogonal resistance mechanisms to occupancy-driven drugs. GRAPHICAL ABSTRACT (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/659082v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1607a40org.highwire.dtl.DTLVardef@fa10deorg.highwire.dtl.DTLVardef@1698232org.highwire.dtl.DTLVardef@1e2e0f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Structural Basis for Cas9-Directed Spacer Acquisition in Type II-A CRISPR-Cas Systems

CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas92-Csn28-Cas18-Cas24) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

molecular biology↗

bioRxiv2025

Dendritic cell-intrinsic TLR/MyD88 signaling promotes the clearance of a live-attenuated oral vaccine

A genital tract pathogenicity-attenuated Chlamydia muridarum mutant, named intrOv, is being developed into an oral vaccine against C. trachomatis infection in the genital tract. Although wild-type C. muridarum persists in the mouse large intestine for long periods, intrOv is cleared by the group 3 innate lymphoid cells that produce IFN{gamma} (IFN{gamma}+ILC3s). The current study aims to reveal the mechanism by which intrOv induces IFN{gamma}+ILC3s for its own clearance from the large intestine. IntrOv-induced IL-23 and IL-23 signaling were required for the clearance of intrOv from the large intestine. The intrOv clearance depended on dendritic cells, as depletion of CD11c+ cells reduced both IL-23 and IFN-{gamma}, resulting in the growth of intrOv. Deletion of MyD88 from dendritic cells, but not phagocytes or epithelial cells, rescued the growth of intrOv, indicating a critical role of MyD88 signaling in dendritic cells. Furthermore, TLR2/4 signaling is also essential for inhibiting intrOv, as the deficiency in TLR2/4 fully rescues the growth of intrOv. Both the TLR and MyD88 signaling must be in the same dendritic cells to inhibit intrOv, as the growth of intrOv in the MyD88- or TLR2/4-deficient mice was blocked by only wild-type bone marrow-derived dendritic cells but not dendritic cells deficient in either MyD88 or TLR2/4. Thus, we have demonstrated a dendritic cell-intrinsic TLR/MyD88/IL-23 pathway for recruiting effectors to clear intrOv from the large intestine. The information may help further improve the efficacy and safety of intrOv and guide the design of future mucosal vaccines.

immunology↗

bioRxiv2025

A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types.

Genome-wide single-cell and spatial long-read approaches have gained traction, but mostly lack single-cell resolution - and yield limited read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation sites from long reads with near-single-cell resolution. Spl-ISO-Seq selects long cDNAs and doubles to triples read lengths compared to standard preparations. Adding a highly specific software tool (Spl-ISOquant) and comparing human post-mortem pre-puberty samples of the visual cortex (8-11 years) to post-puberty samples (16-19 years), we find that cortical layers harbor stronger splicing and poly(A)-site regulation than the adjacent white matter, with enrichment of multiple protein-domain types. For oligodendrocytes however, developmental splicing changes are stronger in white matter. Among cortical layers, layer 4 has the most developmental changes in alternative-exon inclusion in excitatory neurons and in poly(A) sites. We also find many repeat elements, especially ERV1 long terminal repeats downstream of developmentally-regulated layer 4 exons. Overall, alternative splicing changes are linked to synapses - specifically at the post-synapse. Age-linked splicing changes in layers 1-3 and 4 are associated with autism spectrum disorder but not with schizophrenia, amyotrophic lateral sclerosis and Alzheimers disease. These results root developmental splicing changes during puberty and the resulting protein changes in specific layers and cell types. More generally, our new technologies enable new observations for any complex tissue.

genomics↗

bioRxiv2025

A High-Throughput, Flow Cytometry Approach to Measure Phase Behavior and Exchange in Biomolecular Condensates

Biomolecular condensates are essential for cellular organization, yet their formation dynamics and molecular content exchange properties remain poorly understood. In this study, we developed a high-throughput flow cytometry approach to quantify condensate formation, molecular colocalization, and dynamic exchange. Using self-interacting NPM1 condensates as a model system, we benchmarked the use of flow cytometry to broadly characterize their phase behavior across various protein concentrations and salt conditions. We further demonstrated that flow cytometry can assess the colocalization of macromolecules - including antibodies, lipids, small-molecule drugs, and RNAs, within NPM1 condensates. Importantly, we established the first assay to track real-time molecular exchange between preformed condensates and newly added, orthogonally tagged protein. This revealed that condensate aging significantly reduces molecular dynamisms, likely due to altered biophysical properties with time. Compared to conventional imaging techniques, which often require immobilized samples and complex experimental setups, our solution-based approach enables rapid, quantitative analysis of condensate behavior at the single-droplet level. This work provides a powerful framework for studying biomolecular condensates with enhanced precision and scalability, offering new insights into their dynamic properties and molecular interactions.

biophysics↗

bioRxiv2025

Profiling Cytosolic Drug Delivery in Mammalian Cells: A Generalizable Assay for Intracellular Accumulation

The ability of biologically active molecules to access intracellular targets remains a critical barrier in drug development. While assays for measuring cellular uptake exist, they often fail to distinguish between membrane-associated or endosomal trapped compounds and those that successfully reach the cytosol. Here, we present the Chloroalkane HaloTag Azide-based Membrane Penetration (CHAMP) Assay, a novel high-throughput method that employs a minimally disruptive azide tag to report the cytosolic accumulation of diverse molecules in mammalian cells. The CHAMP assay utilizes HaloTag-expressing cells and strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to quantify the presence of azide-tagged test compounds in the cytosol. We demonstrate the versatility of this approach by evaluating the accumulation profiles of small molecules, peptides, and proteins, revealing how structural variations and stereochemical differences influence cytosolic penetration. Our findings with cell-penetrating peptides confirm established structure-activity relationships, with longer polyarginine sequences showing enhanced accumulation. Additionally, we observed that C-terminal amidation and D-amino acid substitutions significantly impact cellular penetration. When applied to supercharged proteins and antibiotics, CHAMP successfully discriminates between compounds with varying accumulation capabilities. This method provides a robust platform for screening cytosolic accumulation while minimizing the confounding effects of large tags on molecular permeability, potentially accelerating the development of therapeutics targeting intracellular pathways.

biochemistry↗

bioRxiv2025

OriGene: A Self-Evolving Virtual Disease Biologist Automating Therapeutic Target Discovery

Here, we present OriGene, a self-evolving multi-agent system that functions as a virtual disease biologist, systematically identifying original and mechanistically grounded therapeutic targets at scale. OriGenes architecture integrates over 600 specialized tools through a Model Context Protocol (MCP), enabling it to reason across diverse data modalities including genomics, protein networks, pharmacology, clinical records and literature evidence, to generate and prioritize target discovery hypotheses. We implemented a strategy combining a knowledge graph-based Tool RAG with an advanced agent selection mechanism to enable dynamic, context-aware tool deployment. Through a self-evolving framework, OriGene continuously integrates human and experimental feedback to iteratively refine its core thinking templates, tool composition, and analytical protocols, thereby enhancing both accuracy and adaptability over time. To comprehensively evaluate its performance, we established TRQA, an original benchmark comprising over 1,900 expert-level question-answer pairs spanning a wide range of diseases and target classes. OriGene consistently outperforms human experts, leading research agents, and state-of-the-art large language models in accuracy, recall, and robustness, particularly under conditions of data sparsity or noise. Critically, OriGene nominated previously underexplored therapeutic targets for liver (GPR160) and colorectal cancer (ARG2), which demonstrated significant anti-tumor activity in patient-derived organoid and tumor fragment models mirroring human clinical exposures. These findings demonstrate OriGenes potential as a scalable and adaptive platform for AI-driven discovery of mechanistically grounded therapeutic targets, offering a new paradigm to accelerate drug development.

bioinformatics↗

bioRxiv2025

TIP-seq: A Single-cell Multiomics Approach for Simultaneous Transcriptome and Intracellular Protein Profiling

Single-cell multiomics technologies have significantly advanced our understanding of cellular heterogeneity; however, the concurrent profiling of mRNA transcripts and target proteins continues to pose a substantial challenge. To bridge the gap between single-cell sequencing and proteomic methods, we developed an innovative single-cell multiomics methodology, termed TIP-seq, which facilitates the simultaneous profiling of the transcriptome and intracellular proteins without prior cell manipulation. In TIP-seq, microfluidic technology is leveraged to integrate oligonucleotide-labeled antibodies with hydrogel beads, allowing for the capture of mRNAs and proteins from individual cells within droplets, thereby enabling high-throughput and precise dual-omics data acquisition. When applied to lung cell lines and non-small cell lung cancer (NSCLC) tissues, TIP-seq analysis revealed notable cellular heterogeneity and molecular dynamics, emphasizing the distinct immune cell interactions within NSCLC tissues. Key immune checkpoint interactions in NSCLC, such as SPP1-CD44, NECTIN2-TIGIT, and NECTIN2-CTLA4, along with functional alterations in tumor-associated dendritic cells (DCs) and T cells, were identified via TIP-seq, underscoring their pivotal roles in mediating immune suppression within the tumor microenvironment. Collectively, TIP-seq represents a powerful methodology for identifying novel therapeutic targets and biomarkers, thereby holding significant potential for the advancement of precision medicine in the treatment of lung cancer and other complex diseases.

biochemistry↗

bioRxiv2025

End Processing in NHEJ by Polymerase {lambda} and PNKP is coordinated during short-range synapsis

Non-homologous end joining (NHEJ) is a major pathway of DNA double strand break (DSB) repair, capable of directly joining both damaged strands of DNA through the coordinated activities of repair factors that detect the termini, physically bridge them together, and perform the chemistry necessary to complete repair. NHEJ is capable of repairing a variety of damaged DNA, employing various accessory end-processing factors to resolve chemically blocked ends, trim overhangs, and fill gaps in order to achieve directly ligatable DNA ends. To investigate the molecular mechanisms underlying end-processing, we determined the cryo-EM structure of the NHEJ specific polymerase Pol {lambda} bound to the short-range synaptic complex, uncovering the mode of its recruitment to the complex as well as a putative model for its activity. Furthermore, the coordinated end-processing activities of the short-range (SR) synaptic complex simultaneously bound by both Pol {lambda} and PNKP, another accessory factor, demonstrates the ability of NHEJ to form large, multifunctional repair complexes capable of processing a variety of different DNA end structures to effect repair.

biophysics↗

bioRxiv2025

Bona fide hematopoietic stem cells in zebrafish originate from the supra-intestinal artery

Hematopoietic stem cells (HSCs) in vertebrates are widely believed to originate from hemogenic endothelial cells (HECs) in the dorsal aorta (DA) through endothelial-to-hematopoietic transition. However, conclusive in vivo DA-specific lineage-tracing studies to confirm this origin are lacking. Here, leveraging infrared light-mediated temporospatial-resolution lineage-tracing technology, we demonstrate that in zebrafish, HSCs do not originate from the DA. Instead, following the DA hematopoiesis, which produces hematopoietic precursors with limited renewal potential, lifelong renewable HSCs emerge from the supra-intestinal artery (SIA). Remarkably, while the DA produces hematopoietic cells from unipotent HECs, the SIA generates hematopoietic cells from both unipotent HECs and bipotent vessel-resident hemangioblasts (VRHs). These VRHs undergo multiple rounds of division and budding, generating both hematopoietic cells and SIA-resident endothelial cells. The distinct characteristics of DA and SIA cells are further reinforced by single-cell transcriptomic profiling. Our findings challenge the current paradigm of HSC development, underscoring the complexity of vertebrate hematopoiesis.

developmental biology↗

bioRxiv2025

The Mycobacterium tuberculosis ESX effector promotes pyroptosis-dependent pathogenicity and dissemination

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/653593v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@8cd5e5org.highwire.dtl.DTLVardef@2ec22forg.highwire.dtl.DTLVardef@1ceb773org.highwire.dtl.DTLVardef@6dfba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic AbstractC_FLOATNO C_FIG Mycobacterium tuberculosis (Mtb) remains a major global health threat, partly due to the extensive cytotoxicity induced during infection. Although GSDMD-mediated excessive pyroptosis promotes pathogen dissemination and tissue damage in tuberculosis, the mechanism remains poorly understood. Here, we identify the effector EccB5, a component of the Mtb ESX-5 secretion system, as a key driver of pyroptosis and hyperinflammatory responses. EccB5 enhances Mtb virulence by inducing pyroptosis of macrophages, promoting bacterial dissemination and exacerbating lung pathology. Conditional knockdown EccB5 increases host cell viability. Mechanistically, EccB5 directly interacts with GSDMD, strengthens its association with caspase-1, and facilitates caspase-1-mediated cleavage of GSDMD both in vitro and in vivo. In summary, our findings uncover a precise mechanism by which Mtb modulates host responses and advances its pathogenicity.

microbiology↗

bioRxiv2025

FASN Inactivation-induced Progranulin (GRN) Expression Promotes Lysosome-dependent Cell Death to Suppress Leukemogenesis

Cancer cells rely on lipogenesis in addition to exogenous lipid uptake, and fatty acid synthase (FASN) is aberrantly overexpressed in myeloid leukemia. However, the precise role of FASN in leukemogenesis in vivo remains elusive. Here, we demonstrated that FASN is essential for leukemogenesis. Ablation of FASN impeded leukemic cell growth, survival, clonogenicity in vitro, and the leukemic burden in vivo. Conditional knockout of FASN barely affected hematopoiesis but significantly attenuated the leukemic progression of MLL-AF9 transplants. By screening a library of platensimycin derivatives, we identified compound MS-C19 as a potent FASN inhibitor. Pharmacological targeting of FASN by MS-C19 suppressed leukemic cell growth and clonogenicity in clinical AML blasts. Mechanistically, both MS-C19 treatment and FASN deficiency triggered the activation of lysosomal and inflammatory gene expression. Loss of FASN led to lysosomal membrane permeabilization and subsequent lysosome-associated cell death, but not lysosome biogenesis. We further identified that GRN, a lysosomal and neuroinflammatory gene, was potently transcribed by TFEB upon FASN loss. Depletion of GRN significantly reversed the inhibitory effects caused by FASN knockdown. Our work demonstrates that FASN is a therapeutic target for myeloid leukemia, and inactivation of FASN by the lead compound MS-C19 provides an alternative approach for leukemic intervention. Key PointsO_LIGenetic ablation of FASN induces apoptosis, impedes leukemia cell growth, and improves the survival of leukemic xenografts. C_LIO_LITargeting FASN by the compound MS-C19, a derivative of the natural microbe antibiotic platensimycin, reduces the cell survival of leukemic cell lines, the clinical leukemic blasts, and MLL-AF9 transformed murine leukemic cells. C_LIO_LIFASN inhibition induces lysosome-dependent cell death that relies on Progranulin (GRN) expression. C_LI

cancer biology↗

bioRxiv2025

Plastic-elastomer heterostructure for robust flexible brain-computer interfaces

Electronics for neural signal recording must be robust across multiple and deep brain regions while preserving tissue-level flexibility to ensure stable tracking over months or years. However, existing electronics cannot simultaneously achieve robustness and tissue-level flexibility, limiting their potential for customizable and scalable neuroscience research and clinical applications. Here, we introduce FlexiSoft, an electronic platform based on a plastic-elastomer heterostructure that uniquely integrates mechanical robustness and tissue-level flexibility. Compared to conventional flexible electronics of similar thickness, the FlexiSoft platform demonstrates an order-of- magnitude improvement in both mechanical robustness (critical energy release rate) and flexibility (flexural rigidity). Leveraging these mechanical advantages, we developed FlexiSoft probe for robust implantation, demonstrated by its ability to withstand repeated insertion and removal, as well as to reach centimeter-scale depths comparable to those in the human brain. The platform enables long-term recording from the same neurons across the hippocampus (HPC) and primary motor cortex (M1) during a months-long motor learning task, thereby revealing long-term dynamic changes in neuronal firing patterns. Additionally, FlexiSofts unique robustness and flexibility enable curved implantation routes, opening new directions of customizable implantation pathways. In summary, we present FlexiSoft as a novel, robust, and tissue-level flexible heterostructure electronics platform that advances flexible brain-computer interfaces (BCIs) with strong translational potential for neuroscience and clinical applications.

bioengineering↗

bioRxiv2025

SCRIPT: predicting single-cell long-range cis-regulation based on pretrained graph attention networks

Single-cell cis-regulatory relationships (CRRs) are essential for deciphering transcriptional regulation and understanding the pathogenic mechanisms of disease-associated non-coding variants. Existing computational methods struggle to accurately predict single-cell CRRs due to inadequately integrating causal biological principles and large-scale single-cell data. Here, we present SCRIPT (Single-cell Cis-regulatory Relationship Identifier based on Pre-Trained graph attention networks) that infers single-cell CRRs from transcriptomic and chromatin accessibility data. SCRIPT incorporates two key innovations: graph causal attention networks supported by empirical CRR evidence, and representation learning enhanced through pretraining on atlas-scale single-cell chromatin accessibility data. Validation using cell-type-specific chromatin contact data demonstrates that SCRIPT achieves a mean AUC of 0.9, significantly outperforming state-of-the-art methods (AUC: 0.68). Notably, SCRIPT obtains a threefold improvement in predicting long-range CRRs (>100 Kb) compared to existing methods. Applying SCRIPT to Alzheimers disease and schizophrenia, we establish a framework for prioritizing disease-causing variants and elucidating their functional effects in a cell-type-specific manner. By uncovering molecular genetic mechanisms undetected by existing computational methods, SCRIPT provides a roadmap for advancing genetic diagnosis and target discovery.

bioinformatics↗

bioRxiv2025

Ciliated cells promote high infectious potential of influenza A virus through the efficient intracellular activation of hemagglutinin

Influenza viruses utilize host proteases to activate the viral fusion protein, hemagglutinin (HA), into its fusion-competent form. Although proteolytic activation of HA is essential for virus replication, the cell-type dependence of HA activation within the airway epithelium and the subcellular location(s) in which it occurs are not well-established. To address these questions, we investigated the proteolytic activation of HA in differentiated human airway epithelial cells using contemporary and historical H1N1 and H3N2 strains. We find that activation is efficient across viral strains and subtypes but depends on cellular tropism, with ciliated cells activating HA more effectively than non-ciliated cells. Similar to prior observations in immortalized cell lines, we find that HA activation occurs intracellularly, constraining the antiviral activity of host-directed protease inhibitors. These results establish that HA activation within the airway epithelium depends on cellular tropism, and identify important considerations for the development of protease inhibitors as antivirals. ImportanceInfluenza entry requires the proteolytic activation of the viral fusion protein, hemagglutinin (HA). Activation occurs as new viruses are produced by infected cells. Efficient proteolytic activation is critical for viral pathogenesis, and inhibiting the requisite proteases could provide an effective host-directed antiviral strategy. To understand cellular constraints on HA activation and its sensitivity to inhibitors, we use complementary approaches to investigate these processes in differentiated airway epithelial cells. We find that ciliated cells activate HA with higher efficiency than non-ciliated cell types, establishing a new mechanism through which cellular tropism and virus infectious potential are connected. We also establish that HA activation begins in the Golgi, which may contribute to the limited the efficacy of inhibitors we observe despite their high in vitro potency.

microbiology↗

bioRxiv2025

FGF21 signals through KLB-expressing glutamatergic neurons in the hindbrain to mediate the effects of dietary protein restriction

Animals adaptively respond to protein restriction by altering both behavior and metabolism. Previous work demonstrated that the metabolic hormone FGF21 acted in the brain to coordinate these adaptive responses, but the exact site of action remains unclear. Here, we identify a discrete population of glutamatergic, Klb-expressing neurons in the nucleus of the solitary tract (NTS), demonstrating that these neurons are key to mediating FGF21 action during protein restriction. Using a novel Klb-Flp mouse line combined with intersectional genetics, we demonstrate that these neurons are directly activated by FGF21. While previous work implicated the SCN, PVN, and VMH in FGF21 action, we find that these areas do not impact the response to protein restriction. Instead, selective ablation of NTS-KLB neurons prevents metabolic adaptations to protein restriction (food intake, food choice, and energy expenditure), while their chemogenetic activation is sufficient to drive these responses. These findings establish NTS-KLB neurons as a critical node for detecting protein status and coordinating whole-body metabolic responses, providing new insight into how the brain monitors and maintains protein homeostasis.

neuroscience↗