Biology subjects

Lan, X.

Publications and source records attributed to Lan, X..

At least 19 recordsLinked to original sources

bioRxiv2026

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

pharmacology and toxicology↗

bioRxiv2026

LoCoFlow: Local-Context Flow Matching for Single-Cell Dynamics and Fate Inference

Single-cell sequencing profiles cellular states at discrete time points, providing population-level snapshots from which developmental and disease-associated trajectories can be reconstructed. Existing optimal-transport and flow-matching approaches typically infer temporal couplings at the level of individual cells, with limited representation of the local manifold geometry of cell-state space and the uncertainty inherent in lineage diversification. We introduce LoCoFlow, a flow-matching framework that combines local point-cloud geometry with conditional fate uncertainty. Overlapping patches define hierarchical transport between populations, and an endpoint-preserving neural bridge regularizes motion and deformation within these patches. Bridge velocities supervise a conditional field with patch-shared and cell-specific latent variables, from which alternative trajectories can be sampled without future observations. Temporal and spatial benchmarks show improvements in transport distance. On lineage-traced LARRY data, LoCoFlow reduces mean raw Brier error from 0.7990 to 0.6620 relative to scDiffEq at the same number of endpoint draws. Expression perturbations further reveal distinct roles for focal-cell and neighborhood information: geometric-patch models show aligned background and whole-patch responses, whereas random-minibatch models show opposing focal and background responses. These findings support local context as a useful representation for probabilistic fate inference and provide testable hypotheses about how it shapes predicted responses.

bioinformatics↗

bioRxiv2026

Multiscale fracture resistance of human annulus fibrosus and its deterioration with degeneration

Soft fibrous tissues must resist fracture under repeated loading, yet how degeneration compromises this resistance remains unclear. We investigated human annulus fibrosus using fracture and fatigue testing, proteoglycan depletion, microscopy, and X-ray scattering. Non-degenerate tissue exhibited pronounced fracture anisotropy, with axial toughness nearly fourfold higher than interlamellar delamination toughness. Degeneration reduced axial and radial toughness despite comparatively preserved tensile modulus and strength. Proteoglycan depletion reduced toughness most strongly in radial and delamination loading. Neither tissue state showed measurable macroscopic crack extension over 20,000 cycles at 70% of its respective monotonic failure strain. Nevertheless, fatigue produced localized structural changes, and degenerate tissue exhibited secondary delamination and diffuse collagen damage. X-ray scattering revealed fatigue- and degeneration-dependent changes in collagen periodicity and intermolecular packing. These findings connect tissue fracture resistance to collagen-matrix interactions across scales and inform the design of crack-resistant biomaterials.

bioengineering↗

bioRxiv2026

Accurate de novo design of peptides from programming biophysical landscape

Peptides regulate virtually every cellular process and emerge as transformative modalities in bioengineering and therapeutics. However, the de novo design of functional peptides remains challenging, as peptide-protein interactions are governed by delicate and dynamic biophysics that are difficult to model, and experimental datasets are scarce. Here we present NeoPep, a generative deep-learning framework that encodes biophysical principles to accurately design functional peptides de novo. By integrating over 5 million peptide-protein complexes spanning experimentally determined, sequence mimics, and structure ensembles, NeoPep learns the complex biophysical landscape governing peptide function and enables its programmable control. In prospective application across 10 diverse and challenging targets, NeoPep generated potent peptide binders, agonists, and antagonists with hit rates of 12.5-66.7%, even in the absence of defined binding sites or structure information. Beyond de novo co-design, NeoPep supports standalone structure or sequence redesign, readily discriminating subtle context differences. In the structure redesign mode, it generates highly selective peptides with atomic-level conformational accuracy (C RMSD < 2.0 [A] to our solved cryo-EM structure). This structural precision circumvents the need for experimental structure determination, further accelerating iterative sequence redesign to yield a 43.3-fold improvement in potency. These findings establish a general framework for translating biophysical principles into actionable peptide functions, with broad implications for basic research, bioengineering, and medicine.

molecular biology↗

bioRxiv2026

Large-scale quantum computing framework enhances drug discovery in multiple stages

Coherent Ising machines (CIMs) excel at solving large-scale combinational optimization problems (COPs), but their insufficient long-term stability has hindered their applications in compute-intensive tasks like computer-aided drug discovery (CADD). By improving fiber vibration isolation and temperature control system, we have implemented a 2000-node CIM named QBoson-CPQC-3Gen achieving stable solutions over one hour on large-scale COPs. Graph-based encoding schemes were further introduced to realize a CIM-based CADD workflow including allosteric site detection, protein-peptide docking and intermolecular similarity calculation. CIM-based methods demonstrated superior speed and accuracy than heuristic algorithms. Especially, QBoson-CPQC-3Gen identified 2 novel druggable sites and bioactive compounds for 6 targets, which were further validated in vitro, in-cell and by crystal structures. Our contributions established a quantum-computing framework for multi-stage drug discovery, representing a significant advancement in both quantum computing applications and pharmaceutical research.

bioinformatics↗

bioRxiv2026

Age-related reconfiguration of locus coeruleus-cortical functional organization

The locus coeruleus (LC) regulates attention, arousal, and adaptive behavior via widespread noradrenergic projections to cortex, yet its large-scale cortical organization and age-related vulnerability remain poorly understood. Using 7T MRI during naturalistic movie-viewing with neutral and negative emotional valence, study-specific LC delineation, and PET-derived receptor-transporter maps, we characterized LC-linked cortical organization in younger and older adults. Functional gradient analysis revealed two dominant axes: a stable primary gradient anchored by catecholaminergic receptor-transporter distributions, and a context-sensitive secondary gradient aligning with the visual-somatomotor (V-S) axis in neutral movie-viewing and the sensorimotor-association (S-A) axis in negative movie-viewing. During negative movie-viewing, older adults exhibited higher global and within-frontoparietal control network dispersion, indicating reduced functional differentiation, a pattern further associated with poorer emotional wellbeing. By revealing chemoarchitectural and context-sensitive LC-linked cortical organization, current work identifies mechanisms underlying age-related LC-linked cortical reorganization with consequences for mental health.

neuroscience↗

bioRxiv2026

Functional connectivity profile of the amygdala subfields associates with emotional well-being in aging

Amygdala-related functional connectivity plays a crucial role in human emotion, cognition, and mental well-being. The amygdala is a highly heterogeneous structure, with subregions that have both distinct and overlapping functions. However, the connectivity patterns of different amygdala subregions--and how these associations vary with age--remain poorly understood. Functional MRI data were analyzed from 68 younger adults and 66 older adults during movie watching in a 7T MRI scanner. Partial least squares (PLS) analysis was used to identify latent variables capturing variance associated with age-related differences in the functional connectivity patterns of three amygdala subregions: the basolateral (BLA), centromedial (CMA), and superficial (SFA) nuclei. In addition, covariance between behavioral measures, such as emotional resilience and cognitive function, and functional connectivity of these subregions was examined. We further explored the associated cognitive processes, correspondence with large-scale brain networks, and the underlying chemoarchitecture of the identified connectivity patterns of the BLA, CMA, and SFA. Multivariate analyses revealed age-related and subregion-specific functional connectivity patterns. Functional connectivity patterns of amygdala subregions were further associated with emotional resilience, which largely overlapped across widespread brain regions; however, their associations differed by age. Stronger coupling of amygdala subregions predicted higher resilience in older adults but lower resilience in younger adults. The identified connectivity patterns were linked to the salience, control, and default mode networks and showed spatial correspondence with mGluR5 and 5-HT6 receptor distributions. These findings highlight age-dependent reorganization of amygdala-related networks that support emotional resilience and provide novel insights into how functional and neurochemical changes of the amygdala subregions contribute to adaptive emotional and cognitive functions in aging.

neuroscience↗

bioRxiv2026

BRD8 Disruption Unlocks Retrotransposon-Driven Tumor-Intrinsic Immunogenicity via Redistribution of Histone Acetylation in Liver Cancer

Overall response rate for immune checkpoint blockade (ICB) therapy remains limited in advanced hepatocellular carcinoma (HCC). Epigenetic perturbation has been shown to enhance tumor-intrinsic immunogenicity and thus reverse resistance to immunotherapy in multiple cancer types. Here, through domain-focused CRISPR/Cas9 genetic screens targeting chromatin regulators, we identified the bromodomain-containing protein 8 (BRD8) as a novel epigenetic factor that functions to suppress tumor-intrinsic immunity through BRD8/EP400 chromatin remodeling complex in HCC. Depletion of BRD8 potently induces anti-tumor immune responses and sensitizes tumors to ICB therapy in mouse tumor models. Mechanistically, BRD8 knockout leads to partial redistribution of H3K27ac to repetitive elements, activating double-stranded RNA (dsRNA) accumulation and subsequent type I interferon (IFN) response, leading to tumor rejection via remodeling the tumor microenvironment. The first-in-class selective chemical probe DN02 targeting the bromodomain of BRD8 also exhibits immunomodulatory and anti-tumor effects. In line with these findings, low BRD8 expression correlated with enriched interferon signatures, elevated immune cell infiltration, and prolonged survival in HCC patients. Together, this study reveals BRD8 as a selective epigenetic vulnerability for promoting tumor immunogenicity, and its bromodomain as a druggable target to overcome immunotherapy resistance in liver cancer patients. SignificanceThe induction of viral mimicry by epigenetic interference could trigger tumor-intrinsic immune response and enhance immunotherapy efficacy. Here, through domain-focused CRISPR/Cas9 screening, we identify BRD8 complex as a regulator involved in the suppression of repetitive elements by controlling H3K27ac distribution and propose BRD8 as a novel target to enhance anti-tumor immunity.

cancer biology↗

bioRxiv2025

NPAS2 attenuates VSMC phenotypic switching in ascending thoracic aortic aneurysm via LPCAT3/PC-PUFA2S-mediated ferroptosis

Ascending thoracic aortic aneurysm (ATAA) is a life-threatening disorder with limited therapeutic options, critically linked to vascular smooth muscle cell (VSMC) phenotypic switching. Neuronal PAS Domain Protein 2 (NPAS2), a circadian rhythm-related transcription factor, regulates diverse physiological processes. However, the biological functions and underlying regulatory mechanisms of NPAS2 in VSMCs during ATAA pathogenesis remain to be elucidated. NPAS2 expression decreased in ATAA patients, PDGF-BB-treated HASMCs and BAPN-administrated mice. NPAS2 depletion facilitated VSMC phenotype switching. VSMC-specific NPAS2 knockout (NPAS2SMKO) mice exhibited aggravated ATAA. Mechanistically, NPAS2 depletion attenuates its transcriptional repression of LPCAT3, subsequently elevating the accumulation of phosphatidylcholines with two polyunsaturated fatty acyl chains (PC-PUFA2S), thereby promoting ferroptosis-induced VSMC phenotype switching and accelerating ATAA progression. Targeting NPAS2 represents a potential therapeutic strategy for ATAA treatment.

molecular biology↗

bioRxiv2025

Precision culturomics enabled by unlabeled single-cell morphology and Raman spectra

Selective enrichment of target bacteria from complex communities such as the human microbiome has remained a challenge. Here we report a solution based on morphology, Raman spectrometry and the Laser-Induced Forward Transfer technology, which works at microbial single cells, many generations before they appear as colonies. We develop a machine learning-based framework that enables species-level targeted sorting of single microbial cells from complex microbiome. We illustrate the utility of this approach in selecting for or against specific bacteria in fecal microbiome samples and the potential for quantifying the molecules expressed based on Raman spectra. Analysis of single-cell cultured genomes reveals that brief antibiotic use drives both pre-existing resistance and de novo mutations in the transpeptidase or efflux pumps of gut commensals, along with convergent evolution between different species. Our precision culturomics method should enable detailed morphological, metabolic, and genomic insights into variations in microbial phenotypes at the single-cell level for microbiome studies.

microbiology↗

bioRxiv2025

Dual function of DOT1L suppresses tumor intrinsic immunogenicity in hepatocellular carcinoma

Immune checkpoint inhibitor (ICB) therapy for many cancers remains limited in patients overall response rate. Discovery and development of more effective combinatorial approaches is urgent. Here, through CRISPR/Cas9 genetic screens, we identify DOT1L as a versatile epigenetic factor that functions to suppress tumor-intrinsic immunity through a dual mechanism. Depletion of DOT1L induces the expression of transposable elements and subsequent type I interferon (IFN) response, and meanwhile lowers ZEB1 levels to further unleash the expression of immune-related genes. In turn, we demonstrate that DOT1L loss or treatment with the clinical stage inhibitor EPZ-5676 sensitizes tumors to ICB with increased immune infiltration in mice. More importantly, EPZ-5676 treatment alone is sufficient to enhance antitumor immunity in humanized mice. TCGA data analysis reveals an inverse correlation between DOT1L expression and IFN signatures across multiple cancer types. These findings provide a rationale for targeting DOT1L to improve tumor immunogenicity and overcome immunotherapy resistance. One Sentence SummaryCRISPR genetic screens identified DOT1L as a potential suppressor of tumor intrinsic immunogenicity

cell biology↗

bioRxiv2025

Key genes and pathways in asparagine metabolism in Alzheimers Disease: a bioinformatics approach

BackgroundAsparagine (Asn) metabolism is essential for maintaining cellular homeostasis and supporting neuronal energy demands. Recent studies have suggested its dysregulation may contribute to Alzheimers disease (AD) pathogenesis; however, the specific genes and regulatory mechanisms involved remain incompletely understood. MethodsFour publicly available microarray datasets (GSE5281, GSE29378, GSE36980, and GSE138260) were utilized to investigate genes with differential expression between control and AD samples. Asparagine metabolism-related genes (AMGs) were retrieved from the GeneCards database, and their intersection with DEGs yielded candidate asparagine metabolism-related differentially expressed genes (AMG-DEGs). Functional enrichment analysis (Gene Set Enrichment Analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes), protein-protein interaction (PPI) network analysis, and centrality scoring identified hub genes. Regulatory mechanisms were investigated through construction of competing endogenous RNA and transcription factor networks. Potential therapeutic compounds were predicted via drug-gene enrichment and evaluated using molecular docking simulations. ResultsThirty-nine AMG-DEGs were identified and found to be enriched in neurodevelopmental, synaptic transmission, and inflammatory signaling pathways. PPI analysis and centrality screening revealed seven hub genes (HPRT1, GAD2, TUBB3, GFAP, CD44, CCL2, and NFKBIA). Regulatory network analysis highlighted specific miRNAs, long non-coding RNAs, and transcription factors involved in their modulation. Drug screening and docking identified Bathocuproine disulfonate, DL-Mevalonic acid, and Phenethyl isothiocyanate as promising compounds with strong binding affinities to hub proteins. ConclusionThis study comprehensively maps the dysregulation of asparagine metabolism in Alzheimers disease and reveals a set of hub genes and regulatory elements potentially involved in disease progression. The predicted therapeutic compounds provide a foundation for further experimental validation and may contribute to the development of novel metabolism-targeted strategies for AD treatment.

neuroscience↗

bioRxiv2025

SIMIT-seq: a bead-free, scalable microfluidic platform for single cell mRNA sequencing with ultrahigh cell-indexing rate

SIMIT-seq is a bead-free, scalable microfluidic platform designed for high-efficiency single-cell mRNA sequencing. Conventional microfluidic-based single-cell RNA sequencing platforms rely heavily on barcoded beads and intricate co-encapsulation schemes, often constrained by double Poisson limitations and the complexities of bead synthesis. In contrast, SIMIT-seq eliminates the need for beads entirely by employing a deterministic, orthogonal barcoding strategy within a two-dimensional micro-well array. This platform achieves an impressive single-cell indexing rate of 96.6% without the need for complex microfluidic operations. Here, we describe the design and fabrication of the SIMIT-seq platform, outline its workflow for transcript capture and library preparation, and demonstrate its application in profiling K562 cells. Our results validate both the high fidelity of molecular barcode immobilization and the systems capability to support downstream single-cell mRNA sequencing. SIMIT-seq offers a cost-effective and scalable alternative for single-cell transcriptomics, providing a promising foundation for future single-cell omics applications.

bioinformatics↗

bioRxiv2025

Compression of functional gradients from rest to naturalistic processing: Moderation effect of age

The functional cortical hierarchy of the human brain, a fundamental principle of brain organization, has been extensively characterized during resting state for healthy younger adults. However, functional re-organization during naturalistic settings, such as movie-watching, and its alterations across the adult lifespan remains poorly understood. Using resting-state and movie fMRI data from two large datasets, Cam-CAN (N=416) and DyNAMiC (N=156), this study conducted a comprehensive comparison of brain organization across two states. We identified a robust reorganization with compression of functional gradients from rest to movie-watching states, which is mediated by changes in functional integration and segregation of brain networks. The extent of compression from rest to movie was significantly greater in older adults and predicted worse cognitive performance among the elderly population. Our findings provide novel insights into how macroscale brain hierarchy is reorganized during naturalistic processing, and how this reorganization during aging impacts cognitive processes, offering a deeper understanding of the neural basis of aging and cognition.

neuroscience↗

bioRxiv2024

Age-related Increase in Locus Coeruleus Activity and Connectivity with Prefrontal Cortex during Ambiguity Processing

Interpreting ambiguous environmental cues, like facial expressions, becomes increasingly challenging with age, especially as cognitive resources decline. Managing these challenges requires adaptive neural mechanisms that are essential for maintaining mental well-being. The locus coeruleus (LC), the brains main norepinephrine source, regulates attention, arousal, and stress response. With extensive cortical connections, the LC supports adapting to cognitive demands and resolving conflicting cues from environment, particularly in later life. Previous research suggests that LC interacts with the prefrontal cortex (PFC) during high-conflict tasks. However, whether LC activity and its connectivity with the PFC support emotional ambiguity processing and contributes to emotional well-being in healthy aging remains unclear. To address this gap, we used 7T-MRI to examine LC function in 75 younger (25.8 {+/-} 4.02years, 35females) and 69 older adults (71.3 {+/-} 4.1 years, 35females) during facial-emotion-recognition task morphed with varying ambiguity: anchor (unambiguous happy or fearful), intermediate-ambiguity (30%happy-70%fearful and 40%happy-60%fearful expressions), and absolute-ambiguity (50%happy-fearful). Behaviorally, participants had longer response times and lower confidence during the absolute-ambiguity condition, while older adults perceived ambiguous faces as happy more frequently than younger adults. Neuroimaging results revealed older adults exhibited greater LC activity and enhanced connectivity with dorsolateral PFC (dlPFC) during absolute-ambiguity compared to younger adults. This heightened connectivity in older adults was linked to better task-independent self-reported mental well-being questionnaires and greater emotional resilience scores derived from principal component analysis. Overall, these findings suggest that greater LC activity supports managing cognitively demanding tasks, while enhanced LC-dlPFC connectivity promotes emotional well-being, highlighting this neural pathways role in healthy aging. Significance StatementUnderstanding how the brain adapts to cognitive and emotional demands with age is key to promoting healthy aging. This study examined whether the locus coeruleus (LC), a brain region critical for regulating attention and arousal, undergoes adaptive changes with age, especially during emotional ambiguity task. Using ultra-high-field imaging, we explored younger and older adults recognize facial expressions with varying ambiguity levels. Our findings indicated that compared to young, older adults showed heightened LC activity and LC-dorsolateral prefrontal cortex (dlPFC) connectivity when processing absolute-ambiguous facial expressions, with enhanced connectivity linked to improved mental well-being. These results suggest higher LC activity supports cognitive demands of ambiguity processing with LC-dlPFC connectivity promoting emotional well-being and resilience, offering insights into mechanisms underlying healthy aging.

neuroscience↗

bioRxiv2024

Single-cell omics for complex microbial communities

Metagenomics has enabled understanding of the microbial composition and functional potential in various environments. Using Laser-Induced Forward Transfer (LIFT) technology, we report high-quality microbial single-cell genome or transcriptome in complex samples such as saliva, mouse gut and human tumor sections. Neighboring cells could be separately retrieved and bacteria could be fluorescently labelled. We illustrate cell-fate commitment with sporulation. The method is scalable and precise, and would empower fundamental insight for microbial populations and single-cell interactions with the host.

microbiology↗

bioRxiv2024

Stable platelet production via the bypass pathway explains the long-term reconstitution capacity of hematopoietic stem cells

Precise understanding how hematopoietic stem cells (HSCs) differentiate in vivo is difficult because we can not trace the in vivo differentiation of HSCs. The single-cell transplantation assay and our own paired daughter cell assay of phenotypic HSCs has revealed the presence of HSCs with reconstitution capacity whose differentiation potential is restricted to the myeloid lineage (MySCs) and the presence of novel direct differentiation pathway form HSCs to MySCs (named myeloid bypass pathway). However, how HSCs differentiate in vivo during hematopoiesis has remained unclear since the paired daughter cell assay was performed partially ex vivo. Aiming to characterize HSCs, including the myeloid bypass pathway, we examined the kinetics of HSC differentiation using a mathematical model. We analyzed data from single-cell transplantation assays in which five blood cell lineages were followed successively after transplantation. An age-related skewing to the myeloid lineage was quantitatively indicated as the production of B cells reduced with age. Dependence on platelet bypass increased with aging and consistently high dependence was associated with long-term reconstitution capacity of the HSCs. Focusing on the ratio of chimerism among cell lineages, the characteristics of dependence on platelet bypass can be accurately determined by the ratio of erythrocyte to platelet chimerism at 8 weeks after transplantation. This new identification criterion is an indicator of long-term reconstitution of HSCs that does not rely on observation of long-term transplantation experiments. These findings reveal the novel characteristics of HSCs related to aging and stemness and highlight the importance of the bypass pathway in HSC differentiation. Significance statementIn vivo differentiation of hematopoietic stem cells (HSCs) is important for understanding blood cell production. Here we investigated the differentiation kinetics of HSCs in single-cell transplantation assays by using a mathematical model. Our findings demonstrate the importance of the bypass pathway in platelet production for the long-term reconstitution capacity of HSCs. These results also suggest the utility of studying time changes in HSC differentiation. The new HSC characteristics and its detection criteria identified in this study are expected to be useful in understanding HSC diversity and aging.

cell biology↗

bioRxiv2024

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↗