Biology subjects

Lee, S. Y.

Publications and source records attributed to Lee, S. Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Noncanonical CD11b on CD8+ T cells promotes cytotoxic effector function

Cancer immunosurveillance largely relies on effector CD8+ T cells, yet surface determinants of cytotoxic competence within tumor-reactive populations remain incompletely defined. Using single-cell surface proteomics in a mouse breast cancer model, we identified a tumor-enriched subset of antigen-experienced CD8+ T cells expressing CD11b, a prototypic myeloid integrin. CD11b CD8 T cells were detected across mouse tumor models and human solid tumors and, within tumor-reactive populations, CD11b marked cells with greater cytotoxic potential. Following activation, naive CD8+ T cells induced ITGAM transcription and CD18-dependent CD11b surface expression. Mechanistically, CD11b localized to the peripheral adhesive ring of the immune synapse and promoted lytic-granule secretion. Accordingly, CD11b+ T cells exhibited enhanced TCR- and CAR-mediated tumor cell killing, whereas CD11b deletion impaired T cell antitumor activity. Together, these findings identify CD11b as an inducible, noncanonical component of the T cell immune synapse that marks cytotoxic competence and promotes antitumor T cell function.

immunology↗

bioRxiv2026

The female larval fly CNS connectome: first draft

We release an enhanced FIB-SEM volume and associated neuron, mitochondria, synapse and glia automated segmentations for the complete, gap- and artifact-free central nervous system of the first instar female larval Drosophila melanogaster. Segmentation data is largely unproofread besides select neurons in the premotor and proprioceptive system and select subvolumes where synapses have been manually annotated, serving as training and evaluation data. The dataset represents the first complete central nervous system of the larval fly, and now awaits substantial improvement of machine vision approaches that effectively leverage domain knowledge for the automated yet data-guided proofreading, or substantial and costly human proofreading. All the data and methods are publicly available for further improvement and analysis.

neuroscience↗

bioRxiv2026

Selective gating of neural modulation through frequency- and behavior-dependent modes during cortical electrical stimulation

Electrical stimulation is widely used to modulate neuronal activity, yet its effects on neuronal circuits in vivo remain poorly understood. This, in turn, has hindered the principled design of stimulation protocols and raised questions about reproducibility that constrain the field's translational impact. Here we combine cortical sinusoidal electrical stimulation (sES) with Neuropixels recordings to characterize stimulation-driven responses in more than 2,700 well-isolated neurons across 53 brain areas in 14 behaving, head-fixed mice. We uncover two distinct, concurrent modes of neural modulation. First is a sustained, brain-wide spike-phase entrainment effect that depends on stimulation frequency: entrainment to slow stimulation is supported by non-synaptic electric field propagation while anatomical connectivity dominates entrainment to higher stimulation frequencies. Second, we find a transient, spatially localized spike-rate modulation mainly mediated through anatomical connectivity that only emerges at high stimulation frequencies by selectively recruiting inhibitory neurons. We show that the two distinct modes are differentially shaped by behavior. By identifying how stimulation frequency governs the mechanism of neural engagement and how behavioral state selectively gates brain-wide entrainment but not local inhibitory recruitment, our results provide a mechanistic foundation for designing targeted, reproducible neuromodulation strategies.

neuroscience↗

bioRxiv2026

An expanded realm of anti-CRISPR-associated proteins and regulatory mechanisms

Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit the CRISPR-Cas immune systems. Rapid acr gene expression upon phage entry enables CRISPR-Cas neutralisation, but can impact phage fitness if unregulated. Therefore, Acr production is often controlled by distinct families of co-encoded anti-CRISPR-associated (Aca) proteins, which are usually helix-turn-helix (HTH) regulators that bind DNA within acr-aca operon promoters. Previously, we demonstrated that the Aca2 family additionally represses Acr production translationally by binding structured RNA motifs within the 5' untranslated regions (UTRs) of the acr-aca mRNA. Here, through systematic bioinformatic analyses, we provide evidence of structured RNA motifs in the 5' UTRs of operons encoding members of other Aca families, and that Aca1 also specifically binds its cognate RNA motif. Additionally, many Aca proteins are predicted to regulate not only their own but also adjacent operons with potential anti-defence genes. Indeed, we show that Aca14, newly identified in this study, represses two predicted anti-defence operons. Aca14 is a ribbon-helix-helix domain protein, revealing regulatory diversity beyond the canonical HTH Aca family members. Collectively, our findings expand the understanding of acr regulation in mobile genetic elements and reveal novel mechanisms by which phages fine-tune anti-defence gene expression. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/740549v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@23e8edorg.highwire.dtl.DTLVardef@19146a8org.highwire.dtl.DTLVardef@b5e553org.highwire.dtl.DTLVardef@1e2ddbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Olfactory Loss Enhances Visual Learning in Drosophila through Structural and Functional Reorganisation

Loss of a sensory modality can enhance performance in the remaining senses. However, the circuit mechanisms by which such cross-modal compensation can improve cognitive functions, including learning, are unknown. Here, we show that compromising olfaction in both larval and adult Drosophila enhances visual associative learning. Using behavioural analysis, functional imaging, and comparative connectomics, we reveal the circuit mechanisms that underlie this improvement. Animals with improved learning ability have enhanced responses to visual stimuli in the higher-order learning circuit. The complementary circuit mechanisms that can enhance these responses are structural reweighting of inputs in the larva, resulting in an increased fraction of synaptic inputs from visual pathways onto neurons in the learning circuit, and a reduction in cross-modal inhibition in the adult. Together, these findings reveal synaptic and disinhibitory circuit mechanisms that enhance learning in higher-order associative networks following sensory loss.

neuroscience↗

bioRxiv2026

Bioactivity-driven discovery of repurposable antivirals as OSCAR inhibitors that promote cartilage protection via transcriptomic reprogramming

Osteoarthritis (OA) is a progressive degenerative joint disorder characterized by cartilage degradation, chronic pain, and impaired joint function. The avascular nature of cartilage isolates chondrocytes from systemic circulation, presenting significant challenges for therapeutic intervention. Despite extensive efforts, no clinically effective disease-modifying osteoarthritis drugs (DMOADs) are currently available. Targeting chondrocyte-specific receptors has therefore emerged as a promising strategy. The osteoclast-associated receptor (OSCAR), expressed on chondrocytes, has been implicated in the regulation of cartilage homeostasis and OA pathogenesis. Here, we applied sBEAR (Structurally similar Bioactive compound Enrichment by Assay Repositioning), a bioactivity-driven virtual screening framework independent of target structural information, to identify small-molecule inhibitors of the OSCAR-collagen interaction. By mining large-scale bioactivity profiles, we identified adefovir (ADV) and brivudine (BRV), as candidate OSCAR inhibitors. Molecular docking analyses indicated that both compounds occupy the collagen-recognition pocket within the OSCAR D2 domain. Intra-articular administration of these compounds in a post-traumatic OA mouse model significantly attenuated OA progression and enhanced chondrocyte regeneration. Both compounds increased Sox9 expression, and transcriptomic analyses revealed that BRV reverses inflammatory and extracellular matrix-degrading transcriptional programs. Together, these findings establish OSCAR as a therapeutically actionable target in OA and highlight ADV and BRV as potential DMOAD candidates.

bioinformatics↗

bioRxiv2025

Holographic fingerprinting reveals oligomer-driven phase separation in Bovine Serum Albumin

Biomolecular condensates formed through phase separation are fundamental to cellular organization. Although the physical principles underlying intrinsically disordered proteins are well understood, the molecular determinants of condensate formation in globular proteins remain elusive. Here, we employ Holographic Particle Characterization, a label-free, high-throughput imaging technique, to investigate the self-assembly of Bovine Serum Albumin (BSA), a model globular protein. We show that this technique reliably differentiates amorphous aggregates from liquid-like condensates by their distinct refractive indices and morphologies. Coupled with size-exclusion chromatography, our analysis reveals that BSA phase separation strictly depends on higher-order oligomeric assemblies. Monomeric and dimeric fractions fail to form condensates under identical crowding conditions. Furthermore, the internal packing density of these condensates is highly tunable via pH-driven protonation changes but remains insensitive to ionic screening. These findings support a model of "emergent multivalency," where oligomerization creates a structural scaffold enabling hydrophobically stabilized phase separation, thereby defining a molecular threshold for globular proteins condensation.

biochemistry↗

bioRxiv2025

The gH/gL/gO Complex of Elephant Endotheliotropic Herpesvirus 1A Functions as a Receptor Binding Complex

Elephant endotheliotropic herpesvirus (EEHV) causes a fatal haemorrhagic disease in young elephants and represents a major threat to the survival of these endangered species. Despite its clinical importance, the mechanisms of EEHV entry and host cell recognition remain poorly understood. Here, we show that the EEHV1A homolog of the Betaherpesvirus glycoprotein O (gO) assembles with glycoproteins H and L (gH/gL) to form a heterotrimeric complex analogous to the human cytomegalovirus (HCMV) gH/gL/gO trimer. Using single-particle cryo-electron microscopy, we determined the structure of the EEHV1A gH/gL/gO complex at 3.2 [A] resolution, revealing a conserved overall architecture but distinct gO topology and disulfide arrangement. The EEHV1A trimer, but not gH/gL alone, bound primary elephant endothelial cells, monocytes, and lymphocytes, indicating that gH/gL/gO serves as the receptor-binding complex of EEHV1A. This binding was specifically blocked by sera from naturally infected elephants, confirming its antigenic relevance. Finally, negative-stain electron microscopy polyclonal epitope mapping of gH/gL/gO-Fab complexes identified a Fab population that bound gO near the apex of the gH/gL/gO trimer, suggesting that these antibodies may interfere with gH/gL/gO-receptor interactions. Together, these findings define the molecular architecture and receptor-binding properties of the EEHV1A gH/gL/gO complex and provide a structural framework for the development of vaccines and antibody-based interventions against EEHV-associated disease.

microbiology↗

bioRxiv2025

Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for single-laser multicolour imaging

Long-Stokes-shift fluorophores enable high sensitivity and multiplexed imaging with single-wavelength excitation. Under single-photon illumination ATTO 490LS exhibits a 165-nm Stokes shift, but its two-photon properties remain uncharacterised. Emission and excitation spectral analyses of ATTO 490LS in ex vivo Drosophila melanogaster brains identified two-photon excitation sensitivity at 940 nm, with peak emission at 640 nm. We demonstrate successful duplexed imaging of ATTO 490LS alongside Alexa Fluor 488 using a single 920-nm fibre laser and dual photomultiplier tubes, enabling distinct measurement of red and green fluorescence signals. These findings establish ATTO 490LS as suitable for multicolour two-photon microscopy with single-laser systems.

neuroscience↗

bioRxiv2025

The phenotypic landscape of the mycobacterial cell

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

microbiology↗

bioRxiv2025

Macrophage-secreted Pyrimidine Metabolites Confer Chemotherapy Resistance in Acute Myeloid Leukemia (AML)

The tumor microenvironment (TME) programs cancer cells to influence therapeutic responses. Macrophages residing in TME switch from pro-phagocytic to tumor-promoting and immunosuppressive phenotypes as cancer develops. While these pro-tumor functions of macrophages are associated with poor outcomes, the underlying mechanisms by which bone-marrow (BM)-associated macrophages fuel myeloid malignancy and their precise contribution to relapse remain undissected. Here, we show expansion of monocyte/macrophage population in leukemia patients post-chemotherapy relapse, and spatial proximity of macrophages to leukemia blasts in the BM niche. This proximity proved functionally consequential--depletion of macrophages delayed leukemia relapse post cytarabine (AraC), a frontline chemotherapy, in patient-derived xenografts (PDX) and syngeneic leukemia models. Mechanistically, a pyrimidine metabolite, deoxycytidine (dC), secreted by BM macrophages, is taken up by leukemia cells to directly inhibit deoxycytidine kinase (DCK) to hamper AraC activation and subsequent resistance in a cell non-autonomous manner. Diagnosis AML patients exhibited significantly higher circulating dC levels than healthy donors, and dC levels further increased following chemotherapy. SAMHD1, which catalyzes deoxynucleoside triphosphates (dNTPs) into deoxynucleoside, was highly abundant in macrophages and mediated dC accumulation. Blockade of dC production in mouse and human macrophages via genetic and pharmacological inhibition of SAMHD1 or DHODH, a critical enzyme in pyrimidine synthesis, restored AraC sensitivity. Combination with DHODH inhibitors significantly delayed AraC relapse in human PDX and mouse syngeneic AML models. Collectively, we identify a metabolic immune-leukemia crosstalk in which SAMHD1high macrophages mediates chemoresistance by secreting pyrimidine metabolites and propose macrophage metabolic reprogramming as a tractable strategy to overcome TME-driven chemoresistance in myeloid leukemia.

cancer biology↗

bioRxiv2025

Beyond Agreement: Standardizing Crowdsourced Synapse Annotations through Proofreading in EM Connectomics

AO_SCPLOWBSTRACTC_SCPLOWReliable synapse identification in volumetric EM is hampered by subtle, 3D cues that yield variable human judgments. We present a standardized proofreading protocol that pairs explicit, operational criteria with machine-learning candidate generation and a two-stage calibration of annotators. In two larval Drosophila melanogaster volumes imaged at 8x8x8 nm, five raters (expert + 4 calibrated annotators) reviewed model-proposed candidates using efficient node-based labels. Multi-rater judgments were aggregated with a probabilistic Dawid-Skene (DS) model to produce consensus labels with calibrated uncertainty. Post-calibration, individual annotator accuracy versus the expert improved (McNemar p < 0.05 for all raters), DS-expert agreement increased, and DS posterior entropy decreased for true positives/negatives, indicating more decisive consensus; gains were modest and dataset-dependent in chance-corrected agreement (Krippendorffs ). By making uncertainty explicit, this protocol converts noisy judgments into auditable supervision suitable for training and evaluation, while honestly communicating residual ambiguity essential for reliable and robust connectomics at scale.

neuroscience↗

bioRxiv2025

A global genetic interaction map of a human cell reveals conserved principles of genetic networks

We generated a genome-scale, genetic interaction network from the analysis of more than 4 million double mutants in the haploid human cell line, HAP1. The network maps [~]90,000 genetic interactions, including thousands of extreme synthetic lethal and genetic suppression interactions. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes, pathways, biological processes, and cellular compartments. Comparative analyses showed that general principles of genetic networks are conserved from yeast to human cells. A genetic interaction network mapped in a single genetic background complements the DepMap gene co-essentiality network, recapitulating many of the same biological connections and also capturing unique functional information to reveal roles of uncharacterized genes and molecular determinants of specific cancer cell line genetic dependencies.

systems biology↗

bioRxiv2025

Modeling Adeno-Associated Viral Vector 6-mediated In Vivo Gene Delivery to Expanded Non-Mobilized Haemopoietic Stem Cells from Transfusion dependent Thalassemia Patients in a Humanized Mouse

Hematopoietic stem cells (HSC) are important targets for gene modification therapies (GMT) as they originate several serious genetic conditions including the {beta}-haemoglobinopathies. Potentially curative ex vivo GMT pose the barriers of accessibility, myeloablation-associated morbidity and prohibitive cost. In vivo GMT using non-integrating single-strand adeno-associated viral vectors (ssAAV) are a promising alternative that address these challenges directly, although the small ssAAV payload limits the capacity to package much larger gene or base editors. We investigated the feasibility of targeting human HSC in vivo with a dual-ssAAV6 strategy, which in future may be useful to deliver split-intein editing tools to overcome this limitation. We engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ humice with human hCD45+CD34+ HSC from transfusion-dependent {beta}-thalassemic patients to test in vivo targeting of hCD45 cells with ssAAV6, then administered 5E+12 genomes/kg of ssAAV6-GFP/ssAAV6-mCherry. Humice showed peak single-transgene expression (GFP+ or mCh+) of 1.96-10.17%, and dual-transgene expression (GFP+mCh+) of 31.77% in circulating hCD45+ cells. Nested hCD45+ from liver, spleen and bone marrow showed single-and dual-transgene expression of 36.13-68.14% and 21.91-59.44% respectively. Secondary transplantation experiments demonstrated long-term persistence of AAV6-transduced hCD45 cells showing single-and dual-transgene expression of 9.19-60.72% and 7.15-9.19% respectively, with significant increase in expression from circulating cells. Minimal pro-inflammatory cytokine expression was observed following ssAAV6 administration in thalassemia humice compared with humice carrying non-thalassemia HSC. Our model demonstrates the efficiency of in vivo ssAAV6-mediated targeting of thalassaemia HSC, potential long-term survivability of transduced cells, and feasibility of a dual-AAV strategy for gene editing, which offers a promising alternative to ex vivo GMT for {beta}-haemoglobinopathies.

molecular biology↗

bioRxiv2025

The unique molecular recognition features of Siglec-10: structural insights into sialoglycan and antibody interactions

Siglec-10 is a sialic acid-binding immunoglobulin-like lectin implicated in immune regulation, yet the molecular basis for ligand recognition and how this is functionally linked to immune modulation remains poorly defined. Herein, we present a multidisciplinary study encompassing structural, biochemical, and cellular approaches to elucidate Siglec-10-carbohydrate interactions and their functional consequences. The crystal structure of the extracellular domain of Siglec-10 in complex with 2-6 sialyllactose revealed the presence of two key arginine residues within the Siglec-10 binding site that interact with the carboxyl group of sialic acid, the canonical R119 and R127, suggesting potential dual contributions to ligand engagement. Saturation Transfer Difference (STD)-Nuclear Magnetic Resonance (NMR) confirmed that R119 is essential for sialoglycan binding in solution, whereas R127 appears dispensable for interactions with glycans under these conditions. In contrast, cell-based binding assays using primary human T cells and engineered monocytic lines demonstrated that both arginine residues (R119 and R127) are critical for cellular recognition, revealing a context-dependent interaction. By obtaining direct images at a molecular resolution of 6-7 nm, super-resolution microscopy further revealed glycan-independent dimerization of the Siglec-10 receptor on the surface of human monocytes. Ligand blockade mediated by anti-Siglec-10 mAb (clone S10A) restores CAR-T cell cytotoxicity in vitro, supporting its role as an immune checkpoint receptor. Finally, although CD24 was not identified as a Siglec-10 ligand on T cells, proximity labeling and mass spectrometry uncovered other sialylated glycoproteins that may mediate this interaction. Together, these results identify Siglec-10 as a modulatory receptor with structural and functional features distinct from other Siglec family members and highlight its potential for therapeutic targeting in cancer immunotherapy.

molecular biology↗

bioRxiv2025

Immortalization and Characterization of Schwann Cell Lines Derived from NF1 Associated Cutaneous Neurofibromas

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition in which patients are heterozygous for a disruptive pathogenic variant in the NF1 gene. The most characteristic feature of the condition NF1 is the neurofibroma, a benign, multi-cellular tumor which initiates when a cell of the Schwann cell lineage gains a somatic pathogenic variant of the other NF1 allele. Neurofibromas developing at nerve termini in the skin are termed "cutaneous" neurofibromas (cNFs), while those developing within larger nerves are termed "plexiform." Most patients develop cNFs beginning in late childhood or early adulthood, continuing throughout life at variable rates. Some patients may develop only a few cNFs, while others suffer from thousands. There are no reliably effective physical or pharmaceutical therapies besides surgical removal. Although these are not life-threatening, they are disfiguring and can interfere with normal life functions. To provide a resource for research, we developed short-term cNF Schwann cell cultures from NF1 patients, from which we subsequently established the first semi-immortalized cNF cell lines through transduction with wild-type human telomerase reverse transcriptase (hTERT) and murine cyclin-dependent kinase 4 (mCdk4) genes. Here we present molecular, cellular, and functional characterization of these cell lines, which will be of utility for investigating and developing NF1 cNF therapies.

genomics↗

bioRxiv2024

Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle

Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.

biochemistry↗

bioRxiv2024

Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex

Short-term facilitation of recurrent excitatory synapses within the cortical network has been proposed to support persistent activity during working memory tasks, yet the underlying mechanisms remain poorly understood. We characterized short-term plasticity at the local excitatory synapses in layer 2/3 of the rat medial prefrontal cortex and studied its presynaptic mechanisms. Low-frequency stimulation induced slowly developing facilitation, whereas high-frequency stimulation initially induced strong depression followed by rapid facilitation. This non-monotonic delayed facilitation after a brief depression resulted from a high vesicular fusion probability and slow activation of Ca2+-dependent vesicle replenishment, which led to the overfilling of release sites beyond their basal occupancy. Pharmacological and gene knockdown (KD) experiments revealed that the facilitation was mediated by phospholipase C/diacylglycerol signaling and synaptotagmin 7 (Syt7). Notably, Syt7 KD abolished facilitation and slowed the refilling rate of vesicles with high fusion probability. Furthermore, Syt7 deficiency in layer 2/3 pyramidal neurons impaired the acquisition of trace fear memory and reduced c-Fos activity. In conclusion, Ca2+- and Syt7-dependent overfilling of release sites mediates synaptic facilitation at layer 2/3 recurrent excitatory synapses and contributes to temporal associative learning.

neuroscience↗