Biology subjects

Lee, S.

Publications and source records attributed to Lee, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Live Spatial Transcriptomics Enables Longitudinal Tissue Mapping

Spatial transcriptomics has become an important technology in biomedical research by preserving the spatial context of gene expression in tissues and enabling the study of cellular states and interactions in situ. However, most existing methods provide only static snapshots of individual tissue specimens, requiring inference of the molecular dynamics underlying development, disease progression, and therapeutic response from different specimens rather than direct tracking in the same living tissue. Here, we introduce Live Spatial Transcriptomics (Live-ST), a technology that enables longitudinal, transcriptome-wide profiling of living tissue. Live-ST combines electroporation-based intracellular sampling and spatial barcoding to enable repeated, spatially resolved mRNA measurements in intact living tissue through next-generation sequencing. We demonstrate its capabilities by (i) benchmarking spatial mRNA capture in patterned two-dimensional cell systems, (ii) spatially profiling transcriptomes in intact, patient-derived living melanoma tissues while preserving tissue viability, and (iii) longitudinally tracking drug-response dynamics within the tumor microenvironment. Together, Live-ST enables repeated, non-destructive measurements of spatial gene expression, providing a new framework for studying dynamic tissue biology in native physiological and pathological contexts.

bioengineering↗

bioRxiv2026

Systematic analysis of genetic vulnerabilities caused by chromosome loss events in cancer

Chromosome loss events are common across tumor types but rarely occur in normal tissue, raising the possibility that these alterations could be exploited as cancer-specific therapeutic vulnerabilities. Here, we integrate genome-scale CRISPR screening data with chromosome-level copy number profiles from 1,150 cancer cell lines and identify 389 dependencies that are selectively associated with individual chromosome loss events. The most significant vulnerabilities uncovered by our analysis are paralogous gene pairs, in which the loss of a paralog on an aneuploid chromosome establishes a unique dependency on its redundant paralog encoded elsewhere in the genome. We experimentally validate two paralog dependencies: loss of chromosome 8p, which encodes the phosphatase PPP2CB, creates a specific dependency on its paralog PPP2CA, while loss of the Y chromosome, which encodes the helicase DDX3Y, creates a specific dependency on its paralog DDX3X. Systematic benchmarking of CRISPR screening data indicates that the selective dependency on DDX3X in loss-of-Y cells exceeds other published aneuploidy-associated vulnerabilities and matches or exceeds most clinically-validated biomarker-target relationships. Furthermore, we found that the acute degradation of DDX3X was sufficient to trigger apoptosis in vitro and tumor regressions in mice. Collectively, these findings establish chromosome loss as a recurrent source of biomarker-defined therapeutic targets, identify paralog insufficiency as a general vulnerability in aneuploid cells, and provide in vivo proof-of-concept for targeting DDX3X in cancers with Y chromosome loss.

cancer biology↗

bioRxiv2026

Isocaloric restriction normalizes body weight but not relative adiposity, adipose inflammation, or gut microbial diversity in high-fat diet-fed mice

Background/Objectives: The separate contributions of excess energy intake and diet composition to high-fat diet (HFD)-induced adiposity, adipose inflammation, and gut microbial changes are unclear. We tested whether restricting HFD intake to the energy intake of low-fat (LF)-fed mice (isocaloric restriction) prevents or reverses these outcomes. Subjects/Methods: Male C57BL/6 mice (n = 8/group) received for 12 weeks an LF diet (LF/LF) or HFD (HF/HF) ad libitum, HFD ad libitum then isocaloric HFD from week 7 (HF/IHF), or isocaloric HFD throughout (IHF/IHF). Adiposity, plasma markers, adipose and ileal gene expression, and fecal microbiota (16S rRNA sequencing, n = 4/group) were assessed. Results: Final body weight in IHF/IHF did not differ from LF/LF (difference 0.1 g, 95% CI -6.5 to 6.7; P > 0.99) and was lower than in HF/HF (-8.2 g, -14.7 to -1.6; P = 0.011). Switching to isocaloric feeding reduced weight gain during weeks 6-12 (HF/IHF vs HF/HF, -6.4 g, -9.8 to -3.1; P < 0.001). IHF/IHF mice had lower glucose (P = 0.031) and cholesterol (P = 0.005) than HF/HF mice. However, visceral fat relative to body weight remained higher than in LF/LF in IHF/IHF (+1.5 percentage points, 0.04 to 2.9; P = 0.042) and HF/IHF (+1.8, 0.4 to 3.3; P = 0.008). Adipose Tnf expression remained elevated in HF/IHF (log2 difference 2.2, 0.2 to 4.2; P = 0.025), and fecal phylogenetic diversity was lower in IHF/IHF than in LF/LF (Faith's PD -3.1, -5.9 to -0.4; P = 0.024). Ileal barrier genes and plasma lipopolysaccharide-binding protein were unchanged. Conclusions: Limiting HFD energy intake normalized body weight, glycemia, and plasma lipids but not relative adiposity, adipose Tnf expression, or the loss of gut microbial diversity. Diet composition affects these outcomes independently of energy intake, so normal body weight does not indicate their reversal.

physiology↗

bioRxiv2026

A Relaxin-Defined Late-Luteal Endocrine Transition State in the Human Menstrual Cycle

Dynamic endocrine transitions govern female physiology, yet several reproductive hormones remain incompletely characterized across the natural menstrual cycle. Relaxin, a peptide hormone classically associated with pregnancy, has recognized vascular, extracellular matrix, and anti-fibrotic actions, but its physiology in nonpregnant women remains poorly defined. We performed dense longitudinal endocrine profiling across 80 natural menstrual cycles from 34 healthy women, complemented by oral contraceptive users, to define circulating relaxin-2 dynamics relative to ovarian steroids. Using generalized additive modeling of cycle-normalized trajectories, we identified relaxin as a highly phase-locked late-luteal hormone with a sharp, reproducible peak distinct from estradiol and progesterone. Progesterone rose earlier after ovulation and peaked at cycle day 23.9, whereas relaxin peaked significantly later at day 28.0, revealing an approximately 4-day temporal offset. Relaxin showed lower inter-cycle variability and narrower temporal dispersion than progesterone, indicating unexpectedly precise endocrine timing. Detectable relaxin persisted into early menses, suggesting continued luteal peptide release across the menstrual boundary. In combined oral contraceptive users, endogenous cyclicity was attenuated and circulating relaxin was effectively absent despite exogenous progestin exposure, implicating ovulation-dependent corpus luteum biology rather than steroid exposure alone as the primary determinant of relaxin secretion. These findings identify relaxin as a reproducible component of normal cyclic endocrine physiology rather than a hormone restricted to pregnancy. The discrete late-luteal relaxin window establishes a previously under-resolved temporal feature of the menstrual cycle and provides a framework for investigating its reproductive, vascular, and connective-tissue significance.

physiology↗

bioRxiv2026

Network Localization of Anxiety as an Early Manifestation of Alzheimer's Disease-related Neurodegeneration

Objective Anxiety is a prominent neuropsychiatric symptom in preclinical Alzheimer's disease (AD), but the neural circuitry linking anxiety and AD remains uncharacterized. We assessed whether anxiety in preclinical AD is independently associated with AD-related neurodegeneration and localizes to a common anxiety network. Methods In cognitively unimpaired (CU) older adults with 3T MRI and amyloid-beta PET (N=1,775), we examined whether an established AD-related atrophy network is associated with anxiety (STAI-6). Then, using Atrophy Network Mapping (ANM), which maps individual cortical atrophy patterns onto a normative functional connectome (N=1000), we derived an anxiety network. We tested its 1) spatial convergence with the AD network and with a causal anxiety network derived from lesion and neuromodulation data, 2) specificity relative to depression, 3) generalizability in samples from independent cohorts with AD and Parkinson's disease (total n=878), and 4) association with longitudinal outcomes. Results In CU older adults, greater AD-network atrophy was associated with higher anxiety, independent of depression, cognition, cortical atrophy, and amyloid burden. Anxiety localized to a network closely resembling the AD network (r=0.74, p=0.04) and the causal anxiety network (r=0.72, p=0.02). This convergence extended across the AD continuum and to the Parkinson's disease cohort and was specific to anxiety relative to depression. Greater anxiety-network atrophy was associated with cognitive decline over 4.6 years. Conclusion Anxiety localizes to a brain circuit resembling the AD-related atrophy network, is consistent across the AD continuum and may extend beyond AD. These findings support anxiety as an independent marker of early AD-related neurodegeneration.

neuroscience↗

bioRxiv2026

Acute social defeat induces cannabinoid type 1 receptor-dependent synaptic depression in the anterior ventral subiculum to anterior hypothalamic nucleus pathway to drive social avoidance in male mice

BACKGROUND: Social avoidance is a protective response that limits exposure to harmful social stimuli. Although avoidance is a characteristic stress phenotype, its circuit-level regulation remains unclear. The ventral subiculum (vSUB), a major hippocampal output, projects to the anterior hypothalamic nucleus (AHN), a key region for stress responses. METHODS: Male mice underwent acute social defeat stress (ASDS) or subthreshold social defeat stress. We combined social interaction, open field, and elevated plus maze testing with optogenetic-assisted slice recording, pharmacology, pathway-specific chemogenetics and optogenetics, retrograde and anterograde transsynaptic tracing, and in situ hybridization. RESULTS: ASDS induced presynaptic depression at anterior vSUB (avSUB) [->] AHN synapses through a glucocorticoid- and cannabinoid type 1 receptor (CB1R)-dependent mechanism, while posterior vSUB (pvSUB) [->] AHN synapses were unaffected. Infusing a CB1R antagonist into the AHN before ASDS attenuated both the synaptic depression and the social avoidance. Chemogenetic activation of the avSUB [->] AHN pathway during ASDS attenuated social avoidance, whereas its inhibition induced avoidance under subthreshold stress. Optogenetic activation of the avSUB [->] AHN pathway produced an immediate increase in social approach. Anterograde tracing revealed that anterior and posterior vSUB neurons project to largely separate AHN populations. The pvSUB [->] AHN pathway modulates anxiety-like behavior without altering social approach. CONCLUSIONS: These findings reveal specialized roles for vSUB subregions in social and emotional responses, and identify presynaptic gating of the avSUB [->] AHN synapse as a control point for stress-induced social withdrawal.

neuroscience↗

bioRxiv2026

Scarless regeneration requires post-translational proteolytic regulation via macrophage-derived Legumain

Macrophages express proteolytic factors in response to injury but how proteases are deployed and regulated during regeneration are incompletely defined. Beyond their transcriptomic expression, activity of individual proteases is defined both by their unique substrate specificity and post-translational modifications of their active forms. Here we show a key role for the lysosomal protease Legumain in controlling ECM dynamics and post-translational processing of the regenerative proteome. Injury interface-targeted Ctsla localization and post-translational processing requires Lgmn in macrophages. Extracellular cues from physical remodeling control differentiation state and injury response of ligament resident fibroblasts. Loss of ECM turnover, including localized Fibronectin 1a/b remodeling, impairs dedifferentiation in legumain mutants and leads to fibrotic scarring. Taken together, we demonstrate a role for macrophage-derived lysosomal proteases as specialized tools required to fine-tune proteome composition and function in regeneration.

cell biology↗

bioRxiv2026

Systematic Functional Interrogation of Neurodevelopmental Disorder Risk Variants

Neurodevelopmental disorders (NDDs) are highly polygenic, yet the functional consequences of most associated non-coding variants remain unclear. We used a massively parallel reporter assay (MPRA) to test 727 NDD-associated non-coding variants in human neural progenitors, identifying 38 expression-modulating variants (emVars) with regulatory activity. These emVars were predicted to alter transcription factor binding motifs linked to neurodevelopmental signaling and cellular metabolism. We mapped emVars to candidate target genes using expression quantitative trait loci and activity-by-contact (ABC) models. We then used CRISPR droplet sequencing (CROP-seq) in human pluripotent stem cell-derived neurons to identify endogenous target genes and characterize their downstream transcriptional effects. CROP-seq preferentially supported ABC predictions, identified additional regulatory relationships, and revealed convergence on developmentally regulated programs involving neuronal connectivity, synaptic function, and mitochondrial biology. Together, these results connect NDD risk variants to endogenous target genes and shared neurodevelopmental transcriptional programs.

genomics↗

bioRxiv2026

Benchmarked sequencing methods reveal hidden mosaic variation in the human brain

Mosaic variants (MVs) record cellular lineages, but their recovery is strongly method-dependent. Here, we benchmarked four duplex sequencing methods alongside bulk and single-neuron genome sequencing in matched human brain tissue. The methods differed in genome representation, MV yield and mutational profiles, with limited caller-level concordance. However, read-level interrogation revealed substantially greater cross-method molecular support. To systematically recover this evidence, we developed Read-level Integration for Variant Evidence Recovery (RIVER), expanding the pool of cross-method-supported candidates by approximately two orders of magnitude relative to caller-level overlap. Orthogonal sequencing at ~10,000x across brain and peripheral tissues validated approximately half of the tested RIVER-rescued candidates. MVs recovered from a single brain region resolved broadly distributed and regionally restricted clones and provided lineage information among individual neurons. These findings show that RIVER substantially expands the pool of endogenous lineage markers available to investigate human development, aging, disease-associated mosaicism and clonal evolution.

genomics↗

bioRxiv2026

The adaptive architecture of tRNA dependencies across physiological tumor environments

The genetic code is decoded by a highly redundant transfer RNA (tRNA) repertoire, yet whether this apparent redundancy serves a functional role beyond ensuring robust translation remains unclear. Here, we establish an atlas of tRNA dependencies through large-scale CRISPRi perturbation mapping across matched in vivo tumor growth and in vitro cell culture for 16 cancer cell lines spanning seven tissue types. During in vivo tumor growth, tRNA dependencies exhibited tissue organization at isodecoder resolution and were associated with codon demand at the isoacceptor level. Strikingly, these relationships were reorganized when cells were cultured in vitro, establishing environmental sensitivity of tRNA dependency--in contrast to protein components of the decoding machinery such as aminoacyl-tRNA synthetases (aaRSs), whose dependencies were largely preserved across environments. Despite pronounced context specificity, environmental alterations in tRNA dependency were dominated by a shared response across cancer cell lines, organized across isoacceptor and isodecoder levels. By disentangling stable cellular identity from environment-associated dependency states, we resolved a shared axis of isoacceptor dependency remodeling and identified specific tRNA isoacceptors associated with promoting or restraining adaptation to the tumor environment. We further connect this functional architecture to differential translation of codon-usage-biased programs associated with proliferation and tumor-microenvironmental stress. Together, our study reveals that tRNA redundancy does not imply functional equivalence, but instead forms a structured and environmentally plastic control layer linking coding information to translational output.

genomics↗

bioRxiv2026

Synthesis-aware generative design in trillion-scale chemical spaces for automated drug discovery

Autonomous drug discovery via generative molecular design is critically bottlenecked by the production of chemically intractable structures. Multi-trillion-scale make-on-demand libraries guarantee synthetic feasibility, but conventional virtual screening cannot efficiently navigate these vast spaces or address multi-parameter optimization. We introduce Hyper Screening X, a generative AI framework integrating structure-based design with automated synthesis hardware. By encoding deterministic reaction logic in a generative flow network, the framework evaluates only 10 million candidates to navigate an 11-trillion-compound space, jointly optimizing physicochemical properties while ensuring compatibility with automated synthesis. Prospective validation against an SLC1A5 variant with a cryptic interface yielded a 96% synthesis success rate and a 50% functional hit rate, identifying two first-in-class lead compounds. This synthesis-aware strategy advances autonomous, closed-loop drug discovery.

bioinformatics↗

bioRxiv2026

TMEM209/NPP-28 partitions PLK-1 between the nuclear envelope and nuclear interior during mitosis

The onset of mitosis in animal cells is marked by nuclear envelope breakdown (NEBD), which requires coordinated disassembly of nuclear pore complexes (NPCs) and the nuclear lamina. Polo-like kinase 1 (Plk1/PLK-1) initiates NEBD through phosphorylation of NPC components, but mechanisms that spatially regulate PLK-1 between NPCs and the nuclear interior are unclear. Here, we identify the Caenorhabditis elegans TMEM209 homolog NPP-28 as a transmembrane nucleoporin that concentrates PLK-1 at the nuclear envelope while limiting its nuclear accumulation to promote NEBD. We identify docking sites within NPP-28 that mediate PLK-1 nuclear envelope-association. NPP-28 is phosphorylated by PLK-1 in vitro, and mutational analysis suggests that NPP-28-mediated docking facilitates NEBD, whereas phosphorylation prevents premature NEBD. Simultaneous loss of npp-28 and the transmembrane nucleoporin npp-12/gp210 exacerbates mitotic NEBD defects in early embryos and causes severe defects in germline development. Together, our findings identify NPP-28 as a spatial regulator of PLK-1 during mitosis with functional relationships with other conserved transmembrane nucleoporins depending on context.

cell biology↗

bioRxiv2026

TangentXY: Improved copy-number estimation of sex chromosomes reveals features associated with aneuploidies in chromosomes X and Y across cancers

Signal-to-noise ratios in copy-number estimates across genomic loci improve with the use of large panels of germline reference data. However, in the case of sex chromosomes, germline samples differ between males and females (males have one X chromosome (chrX) and one Y chromosome (chrY), while females have two chrXs), limiting the size of the reference sets to those that match the sex of the index sample. Frequent mosaic loss of chrY in normal male tissue also leads to biases when not accounted for. Here we report TangentXY, a copy-number normalization method that uses both male and female reference samples, correcting for sex differences in chrX and accounting for chrY mosaicism, to assess sex chromosome copy numbers for samples of either sex. We show that TangentXY maintains sex-specific differences in chrX and chrY while minimizing noise. We also used TangentXY to analyze associations between copy-number alterations in sex chromosomes and cancer characteristics, and found that losses in the chrX but not chrY are correlated with TP53 mutation, that gain of chrX can predict patient prognosis in certain tumor types, and that the vast majority of gains of chrX in males remain active, whereas gains of chrX in females are divided approximately equally between active and inactive chrXs. We also found that loss of chrY correlates with inactivating driver mutations in escape genes in chrX, suggesting a mechanism by which its frequent loss promotes cancers in males.

cancer biology↗

bioRxiv2026

Temporal control of mitochondrial mutagenesis reveals the fate of mtDNA mutations with age

Mutations in the mitochondrial genome (mtDNA) play a critical role in the aging process and a wide variety of age-related diseases. However, it remains unclear when the mutations that drive physiological decline arise. To answer this question, we generated a new mouse model in which mitochondrial mutagenesis can be confined to a defined window of time. Surprisingly, we found that mutations that arise during the first two months of life are sufficient to drive a wide variety of age-related pathologies, and that the severity of this pathology is broadly regulated by distinct, tissue-specific selective pressures that control the fate of mtDNA mutations with age. Further, we found that selection against deleterious variants can be modulated by manipulation of mitochondrial fusion in vitro and in vivo. These observations raise the possibility that in some tissues, the pace of aging is pre-determined by events that occur early in life and that interventions targeting mitochondrial fusion may be able to slow down or reverse the expansion of these pathogenic variants. These results carry far-reaching implications for strategies aimed at preventing or delaying age-related decline.

genetics↗

bioRxiv2026

fastACCORD enables ultrahigh-dimensional partial correlation modeling for multi-omic data integration

Gene co-expression networks reflect transcription factor-associated regulation together with epigenetic influences such as DNA methylation, chromatin states, and histone modifications. To distinguish gene-gene dependencies that persist after accounting for methylation covariation, large-scale statistical inference conditioning on hundreds of thousands of molecular features is necessary, but the task remains computationally intractable for conventional Gaussian graphical modelling approaches. Here we present fastACCORD, a scalable computational framework for ultrahigh-dimensional partial correlation modeling. fastACCORD combines row-separable optimization, {ell}2 stabilization, and a semismooth Newton solver in a PyTorch implementation for CPU and CUDA-enabled GPU hardware. We applied it to matched transcriptomic and methylomic profiles from 16 TCGA cancer types and obtained joint networks containing >300,000 molecular features per cancer. The resulting multi-omic networks revealed cancer-specific methylation-expression dependencies, including recurrent methylation-associated expression repression of metabolic genes. Methylation-adjusted gene co-expression networks were sparser than networks estimated from mRNA data alone, but more enriched for ChIP-seq-supported TF-target relationships and curated co-regulon annotations. TF-target subnetworks further revealed cancer-specific architectures consistent with lineage identity, oncofetal reactivation, and tumor microenvironment-associated programs. Together, these analyses establish fastACCORD as a practical framework for ultrahigh-dimensional multi-omic partial correlation modeling and demonstrate how joint modeling of transcriptomic and epigenomic measurements can refine the interpretation of gene co-expression networks.

bioinformatics↗

bioRxiv2026

Pathogenic mutations in ATAD3A cause dysregulation of RagC/D-TFEB axis and disrupt lysosomal homeostasis

We previously discovered that a de novo variant p.R528W in ATAD3A, encoding a mitochondrial membrane-anchored protein, causes a human neurological syndrome. While ATAD3A mutations induce aberrant lysosomal expansion accompanied by undigested material in the lysosomes, how mutant ATAD3A disrupts lysosomal homeostasis and whether this contributes to neurodevelopmental defects remain unknown. Here we show that pathogenic ATAD3A p.R528W expression disrupts the mTORC1-TFEB axis as revealed by dysregulation of mTORC1 substrate phosphorylation, TFEB nuclear localization, and CLEAR gene activation associated with lysosomal biogenesis. ATAD3A binds to lysosome-localized Rag C/D GTPases, which constitute a platform for TFEB recruitment, with pathogenic variants increasing this association and thereby decreasing lysosomal localization of Rag GTPases. Importantly, overexpression of RagC or RagD restores TFEB phosphorylation in human cells expressing p.R528W, and RagC-D overexpression or TFEB/Mitf knockdown rescues lysosomal expansion and neurodevelopmental defects in Drosophila. These data indicate that disrupted Rag GTPase recruitment to lysosomes and subsequent aberrant TFEB/Mitf activation contribute to neurodevelopmental and lysosomal phenotypes caused by pathogenic mutations in ATAD3A. Our work reveals a novel role for the mitochondrial resident protein ATAD3A in modulating lysosomal homeostasis through regulation of the mTORC1-TFEB axis, providing a mechanistic link between impaired mitochondrial and lysosomal homeostasis in a neurodevelopmental disorder.

genetics↗

bioRxiv2026

taxutils: Tools for managing and operating on NCBI taxonomy and accession mappings

Research in bioinformatics demands that many workflows and operations be consistently repeated across different tools. These operations include managing a taxdump, dealing with accession mappings, and performing analysis on the taxonomic tree. We introduce taxutils to address and alleviate the strain of these common workflows. To exhibit the extent of the package's capabilities, we introduce a strategy for assessing quality of an assembly via its potential impact on and perturbation of its mapping to a lowest-common-ancestor index. We illustrate the steps required for such an analysis, and show how the tool makes data management easy and otherwise-complex workflows simple.

genomics↗

bioRxiv2026

Human iPSC-derived neurons identify West Nile virus neuropathogenesis signatures and reveal strain-dependent neuroattenuation

West Nile virus (WNV) is one of the leading causes of arthropod-transmitted encephalitis in the world, yet its effects on neurons, the principal target of WNV in the brain, remain poorly defined. Here we establish and use Ngn2-induced human iPSC-derived glutamatergic neurons to define the neuronal response to WNV through integrated temporal proteomics and transcriptomics. Temporal multi-omic profiling revealed that infection activated innate immunity and the Unfolded Protein Response while suppressing axonogenesis and synaptic programs. WNV infection also disrupted calcium homeostasis, reduced STMN2 abundance, and promoted cytoplasmic redistribution of TDP-43. Applying this neuronal model to characterize contemporary California isolates, we identified LA24, a lineage 1 strain that replicates efficiently in non-neuronal cell lines but is selectively attenuated in neurons, with reduced viral burden in the brain and attenuated pathogenicity in mice. Together, these findings uncover neuron-specific signatures of WNV pathogenesis and demonstrate that neuronal replication phenotypes can reveal strain-dependent virulence differences not captured by conventional cell lines.

microbiology↗