Biology subjects

Lee, T.

Publications and source records attributed to Lee, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Oxidative-stress related increase in keratoconus tear MDA and GPX3 while NRF2-antioxidant functions decrease in stromal cells

Keratoconus (KC) is a common eye disease where the cornea undergoes degenerative thinning and steepening. The absence of biomarkers for early diagnosis prior to the onset of overt corneal phenotypes and the lack of curative treatments rooted in a fundamental understanding of KC biology remain significant challenges. To address these issues, we investigated the role of unresolved oxidative stress in KC pathogenesis. Malondialdehyde (MDA) a lipid peroxidation byproduct that accumulates during oxidative stress was significantly elevated in the tears of KC patients compared to unaffected controls and positively correlated with maximal keratometry (Kmax), a measure of KC severity. Similarly, the secreted antioxidant glutathione peroxidase 3 (GPX3), was significantly increased in patient tears, and strongly correlated with Kmax. In a cell culture model of oxidative stress, KC corneal stromal cells displayed increased apoptosis and suboptimal activation of NRF2, a transcription factor master regulator of antioxidant genes. Conversely, inhibition of NRF2 in donor stromal cells elicited KC-like cellular phenotype, whereas sulforaphane, an NRF2 booster restored antioxidant gene expression and the deposition of cornea-typical collagens. Our study identified cellular antioxidant signaling dysregulations in keratoconus where sulforaphane treatment may be restorative. Consistent increases in patient tear MDA and GPX3 present these as promising biomarkers for KC diagnosis and severity predictions.

cell biology↗

bioRxiv2025

Thalamofrontal synaptic weakening underlies short-term memory deficits from adolescent NMDAR hypofunction

N-methyl-D-aspartate receptor (NMDAR) hypofunction is linked to schizophrenia, but prevailing models centered on reduced number and activity of inhibitory interneurons, and the resulting excitation-inhibition (E-I) imbalance, do not explain selective cognitive impairments. Here, we show that repeated adolescent NMDAR antagonism produces short-term memory (STM) deficits by weakening thalamofrontal (TF) synaptic transmission. In mice repeatedly exposed to ketamine, STM impairment coincided with reduced release probability and vesicle refilling at mediodorsal thalamus (MD) [->] dorsomedial prefrontal cortex (dmPFC) synapses, without detectable changes in layer 2/3 [->] layer 2/3 synaptic release probability, intrinsic excitability, or synaptic ultrastructure. These presynaptic deficits were accompanied by diminished direction-selective population coding in the dmPFC, as revealed by decoding analysis, and impaired delayed alternation performance in the Y-maze. Chemogenetic strengthening of MD [->] dmPFC projections restored both neural selectivity and behavior. Our findings identify a circuit-specific presynaptic mechanism linking adolescent NMDAR hypofunction to cognitive dysfunction, challenging interneuron-centric models and establishing TF synapses as a potential therapeutic target in NMDAR-related disorders.

neuroscience↗

bioRxiv2025

CCAAT-enhancer binding protein delta functions as a tumor suppressor gene in acute myeloid leukemia

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

cancer biology↗

bioRxiv2025

Toggling of NKG2A expression drives functional specialization of iPSC-derived CAR NK cells

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of {beta}2-microglobulin (B2) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-{gamma} production and cytotoxic activity against target cells with forced high expression of single-chain {beta}2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

immunology↗

bioRxiv2025

Dual-encoder contrastive learning accelerates enzyme discovery

The ability to engineer enzymes for desired reactions is a cornerstone of modern biotechnology, yet identifying suitable starting proteins remains a critical bottleneck. Although contrastive learning offers a compelling computational approach for enzyme discovery, these models have yet to be implemented at scale or proven effective in real-world experimental settings. Here, we present Horizyn-1, a computationally efficient deep learning framework that enables large-scale reaction-to-enzyme recommendation validated through comprehensive experimental testing. Leveraging a combination of reaction fingerprints and protein language models, we trained Horizyn-1 on millions of reaction-enzyme pairs to achieve state-of-the-art performance, recovering an enzyme with correct activity within the top 100 hits for over 75% of test reactions. We experimentally validate Horizyn-1 across three enzyme discovery scenarios: identifying enzymes for orphan reactions, predicting enzyme promiscuity for both characterized and uncharacterized enzymes, and discovering enzymes for non-natural biochemical reactions including lysine-driven transaminations that enable efficient synthesis of non-canonical amino acids. On underrepresented reaction classes, we find that fine-tuning with fewer than 10 additional reactions can dramatically improve performance. Furthermore, a logarithmic scaling of model performance with training dataset size suggests continued improvement with larger and more diverse reaction datasets. Horizyn-1 addresses the critical bottleneck of sourcing initial enzymes for optimization campaigns, enabling efficient and scalable in silico screening for enzymes with desired activities and promising to accelerate future efforts in biocatalysis and metabolic engineering.

biochemistry↗

bioRxiv2025

An image-based transcriptomics atlas reveals the regional and microbiota-dependent molecular, cellular, and spatial structure of the murine gut

The gastrointestinal environment is home to a massive diversity of diet-, host-, and microbiota-derived small molecules, collectively sensed by a remarkable variety of cells. To explore the cellular and spatial organization of sensation, we used MERFISH to profile receptor expression across 2.1 million cells in multiple regions of the murine gut under specific-pathogen-free (SPF) and germ-free (GF) conditions. This atlas revealed expected and novel cell types--including a candidate murine homolog of human BEST4 enterocytes--demonstrated cell-type regional specialization, discovered extensive location-dependent spatial fine-tuning in mucosal cell expression, and suggested cell-type specific mediators of the effects of microbiota-derived small molecules. In addition, this atlas revealed that, aside from immune cell abundance, many aspects of the murine gut are host-intrinsic and modified only modestly in the absence of a microbiota. Collectively, this atlas provides a valuable resource for understanding the cellular and spatial organization underlying small molecule sensation in the gut.

genomics↗

bioRxiv2025

Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

Fractures heal by rapid formation of mineralized callus. Essential to this process is proliferation of periosteal cells to supply bone-forming osteoblasts. To better understand the role of cell proliferation in fracture healing, we asked: How is callus composition altered when osteolineage cells proliferation is blocked? Do mature osteoblasts proliferate to contribute to callus formation? First, mice expressing herpes simplex virus-thymidine kinase (HSV-TK) driven by the 3.6Col1a1 promoter were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Analysis of callus cells using single-cell RNA-seq revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than control mice, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells that expressed the early osteoblast markers osterix (OSX) and the late marker osteocalcin (OCN) were in the cell cycle. Next, we targeted proliferating osteoblasts at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with novel ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited poorer radiographic healing, decreased callus bone volume and a shift from callus bone to fibrous tissue. We conclude that osteoblasts, often considered post-mitotic, proliferate after fracture to contribute to formation of mineralized callus essential to healing.

physiology↗

bioRxiv2025

Joint Modeling of Longitudinal Biomarker and Survival Outcomes with the Presence of Competing Risk in Nested Case-Control Studies with Application to the TEDDY Microbiome Dataset

MotivationLarge-scale prospective cohort studies collect longitudinal biospecimens alongside time-to-event outcomes to investigate biomarker dynamics in relation to disease risk. The nested case-control (NCC) design provides a cost-effective alternative to full cohort biomarker studies while preserving statistical efficiency. Despite advances in joint modeling for longitudinal and time-to-event outcomes, few approaches address the unique challenges posed by NCC sampling, non-normally distributed biomarkers, and competing survival outcomes. ResultsMotivated by the TEDDY study, we propose "JM-NCC", a joint modeling framework designed for NCC studies with competing events. It integrates a generalized linear mixed-effects model for potentially non-normally distributed biomarkers with a cause-specific hazard model for competing risks. Two estimation methods are developed. fJM-NCC leverages NCC sub-cohort longitudinal biomarker data and full cohort survival and clinical metadata, while wJM-NCC uses only NCC sub-cohort data. Both simulation studies and an application to TEDDY microbiome dataset demonstrate the robustness and efficiency of the proposed methods.

bioinformatics↗

bioRxiv2025

Deviant functional connectivity patterns in the EEG and their potential use for dyslexia screening

Developmental dyslexia (DD) is a common learning disorder with potential neural origins. While EEG-based brain activation measures combined with machine learning have shown promise for DD screening, these approaches often lack validation on independent participants, which is a crucial step for practical application. This study developed an EEG-based screening approach and investigated the neural correlates of DD in Chinese children. EEG signals were recorded from 130 children (82 with DD, 48 typically developing) aged betweeen 7 and 11 during resting-state and working memory tasks. The EEG data were preprocessed into clean segments to compute functional connectivity (FC) matrices across four frequency bands (delta, theta, alpha, beta). The segments were split into two independent samples, ensuring independence at the participant level: Sample 1, used for training and five-fold cross validation of the convolutional neural networks, and Sample 2, used for cross-sample validation with the trained model. The beta-band FC index in the eyes-open condition achieved the highest within-sample classification accuracy (98%) and cross-sample accuracy (70%, p < .001). Discriminative FC patterns revealed that children with DD exhibited reduced temporal-parietal and central connectivity but increased frontal-central connectivity, likely reflecting compensatory mechanisms. Within the DD group, stronger FCs showed significant negative correlations with Chinese word reading accuracy and fluency. These findings suggest that EEG-based FC measures can effectively distinguish DD and reveal neural markers associated with impaired reading performance. This approach shows promise for noninvasive screening and deeper insight into the neural basis of DD, particularly in non-alphabetic language systems.

neuroscience↗

bioRxiv2025

Perilysosomal Ca2+ overload impairs autophagic degradation in β-cell lipotoxicity

Saturated fatty acids impose lipotoxic stress on pancreatic {beta}-cells, leading to {beta}-cell failure and diabetes. In this study, we investigate the critical role of organellar Ca2+ disturbance on defective autophagy and {beta}-cell lipotoxicity. Palmitate, a saturated fatty acid, induced perilysosomal Ca2+ elevation, sustained mTORC1 activation on the lysosomal membrane, suppression of the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel, and accumulation of undigested autophagosomes in {beta}-cells. These Ca2+ aberrations with autophagy defects by palmitate were prevented by a mTORC1 inhibitor or a mitochondrial superoxide scavenger. To alleviate perilysosomal Ca2+ overload, strategies such as lowering extracellular Ca2+, employing voltage-gated Ca2+ channel blocker or ATP-sensitive K+ channel opener effectively abrogated mTORC1 activation and preserved autophagy. Furthermore, redirecting perilysosomal Ca2+ into the endoplasmic reticulum (ER) with an ER Ca2+ ATPase activator, restores TRPML1 activity, promotes autophagic flux, and improves survival of {beta}-cells exposed to palmitate-induced lipotoxicity. Our findings suggest oxidative stress-Ca2+ overload-mTORC1 pathway involves in TRPML1 suppression and defective autophagy during {beta}-cell lipotoxicity. Restoring perilysosomal Ca2+ homeostasis emerges as a promising therapeutic strategy for metabolic diseases.

physiology↗

bioRxiv2025

The Single Cell Landscape of the Human Vein After Arteriovenous Fistula Creation and Implications for Maturation Failure

The biological mechanisms underlying arteriovenous fistula (AVF) maturation in hemodialysis patients remain poorly understood despite decades of research. To address this gap, we investigated the cellular changes in the venous wall after fistula creation in histological biopsies of longitudinal veins and AVF samples (N=23 patients). Using single-cell RNA sequencing of 70,281 cells from pre-access veins, mature, and failed AVFs (N=20 patients), we created a complementary transcriptomic atlas of the human vein before and after anastomosis. Postoperatively, the fistula exhibited increased intimal hyperplasia and cell number but reduced cell density, indicating that extracellular matrix (ECM) deposition was more prominent than cell accumulation. Analysis of 14,475 cells from fistulas obtained within one week of creation revealed that inflammation drives early adaptation across all vascular cell types. This includes the pro-inflammatory activation of endothelial cells (ECs) and production of a hyaluronic acid-rich neointima by fibroblasts. By 13 {+/-} 6 weeks, transcriptomic profiles continue to reflect active healing of the vasculature by ECM-producing myofibroblasts and fibroblasts that were found localized throughout the vascular wall, including the intima, using immunofluorescence and in-situ hybridization. Postoperative ECs maintained significant hemostatic adaptations and upregulation of inflammatory molecules (ACKR3, ICAM1, IL1R1, COL8A1) supporting their role as gatekeepers of immune cell infiltration. Comparative analyses of failed versus mature AVFs revealed persistent inflammatory signaling among macrophages, ECs, myofibroblasts, and fibroblasts in association with AVF failure. These findings uncover previously unrecognized cellular and molecular patterns in human veins following AVF creation, providing novel insights and potential therapeutic targets to improve AVF outcomes. TRANSLATIONAL STATEMENTArteriovenous fistulas (AVF) are a special type of blood vessels that provides access to a patients bloodstream during hemodialysis treatments. The AVF is surgically created by connecting an artery and a vein, after which the vein heals and enlarges in a process called "maturation". We do not fully understand how maturation occurs. This prevents us from designing therapies that ensure proper enlargement of the AVF, which fails in up to 40% of patients. This study investigates how a vein transforms into an AVF in 43 patients with end-stage kidney disease undergoing surgery for AVF creation. We analyze the modifications of structural components of the vein and of diverse populations of cells that direct healing and maturation. Our findings suggest that, contrary to current beliefs, the best therapies to improve AVF maturation should target the accumulation of non-cellular components in the vein and the inflammatory factors that trigger such accumulation.

molecular biology↗

bioRxiv2025

Tissue-dependent mechanosensing by cells derived from human tumors

Alterations of the extracellular matrix (ECM), including both mechanical (such as stiffening of the ECM) and chemical (such as variation of adhesion proteins and deposition of hyaluronic acid (HA)) changes, in malignant tissues have been shown to mediate tumor progression. To survey how cells from different tissue types respond to various changes in ECM mechanics and composition, we measured physical characteristics (adherent area, shape, cell stiffness, and cell speed) of 25 cancer and 5 non-tumorigenic cell lines on 7 different substrate conditions. Our results indicate substantial heterogeneity in how cell mechanics changes within and across tissue types in response to mechanosensitive and chemosensitive changes in ECM. The analysis also underscores the role of HA in ECM with some cell lines showing changes in cell mechanics in response to presence of HA in soft substrate that are similar to those observed on stiff substrate. This pan-cancer investigation also highlights the importance of tissue-type and cell line specificity for inferences made based on comparison between physical properties of cancer and normal cells. Lastly, using unsupervised machine learning, we identify phenotypic classes that characterize the physical plasticity, i.e. the distribution of physical feature values attainable, of a particular cell type in response to different ECM-based conditions.

biophysics↗

bioRxiv2024

PrimalScheme: open-source community resources for low-cost viral genome sequencing

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, Covid-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses, and has generated over 67,000 primer schemes for the global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online [~]250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primer schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.

genomics↗

bioRxiv2024

How does Gloger's rule predict near- and mid-infrared radiation in birds?

Animal coloration has diverse functions such as camouflage, communication, thermoregulation, protection from UV damage and more, and can be shaped by environmental selective pressures. Some climactic selective pressures are strong enough to produce consistent patterns in many species across large-scale geographic gradients. One pattern in endothermic animals is Glogers rule, which predicts that populations in hot, humid areas will be darker than those in cool, dry areas. This pattern has been demonstrated in several species across latitudinal gradients and is expected to relate to the selective effects of both local thermoregulatory pressures and humidity. However, shortwave radiation from sunlight extends beyond the visible spectrum [400-700 nm] into the near-infrared; thus, thermal pressures often result in changes in surface reflectance characteristics beyond the visible [e.g., 700-2500 nm]. Further, heat exchange with the environment extends into the mid-infrared, including MIR heat loss through the atmospheric transmission window [7.5 - 14 um]. Here, we examine both UV-NIR absorption and MIR emittance in five species of birds that have been shown to follow, or not follow, Glogers rule. We show that NIR absorption varies by species and population in ways that correspond to their habitat and thermoregulatory strategies. MIR emittance, by contrast, was very stable across both species and populations but differed across populations of Northern Bobwhites. We conclude by highlighting the importance of extending coloration research into the NIR and MIR. Further consideration of infrared radiation is necessary for a complete view of animals phenotypic diversity and possible responses to thermal challenge.

ecology↗

bioRxiv2024

Epigenetic control of Topoisomerase 1 activity presents a cancer vulnerability

DNA transactions introduce torsional constraints that pose an inherent risk to genome integrity. While topoisomerase 1 (TOP1) activity is essential for removing DNA supercoiling, aberrant stabilization of TOP1:DNA cleavage complexes (TOP1ccs) can result in cytotoxic DNA lesions. What protects genomic hot spots of topological stress from aberrant TOP1 activity remains unknown. Here, we identify chromatin context as an essential means to coordinate TOP1cc resolution. Through its ability to bind poly(ADP-ribose) (PAR), a protein modification required for TOP1cc repair, the histone variant macroH2A1.1 establishes a TOP1-permissive chromatin environment, while the alternatively spliced macroH2A1.2 isoform is unable to bind PAR or protect from TOP1ccs. By visualizing transcription-induced topological stress in single cells, we find that macroH2A1.1 facilitates PAR-dependent recruitment of the TOP1cc repair effector XRCC1 to protect from ssDNA damage. Impaired macroH2A1.1 splicing, a frequent cancer feature, was predictive of increased sensitivity to TOP1 poisons in a pharmaco-genomic screen in breast cancer cells, and macroH2A1.1 inactivation mirrored this effect. Consistent with this, low macroH2A1.1 expression correlated with improved survival in cancer patients treated with TOP1 inhibitors. We propose that macroH2A1 alternative splicing serves as an epigenetic modulator of TOP1-associated genome maintenance and a potential cancer vulnerability.

molecular biology↗

bioRxiv2024

Detection of a reassortant swine- and human-origin H3N2 influenza A virus in farmed mink in British Columbia, Canada

In December 2021, influenza A viruses (IAV) were detected in a population of farmed mink in British Columbia, Canada. Based on genomic sequencing and phylogenetic analysis, these IAVs were subtyped as H3N2s that originated from reassortment of swine H3N2 (clade 1990.4h), human seasonal H1N1 (pdm09), and swine H1N2 (clade 1A.1.1.3). This reassortant has been subsequently observed in swine in several Midwest American states, as well as in swine and turkeys in Ontario, suggesting its spillover into farmed mink in British Columbia was incidental to its broader dissemination in North American swine populations. These detections reaffirm the need for extensive genomic surveillance of IAVs in swine populations to monitor reassortments that might become public health concerns. They also highlight the need for closer surveillance of IAVs in mink to preserve animal health, protect agricultural interests, and monitor potential zoonotic threats.

microbiology↗

bioRxiv2024

Chemical codes promote selective compartmentalization of proteins

Cells have evolved mechanisms to distribute [~]10 billion protein molecules to subcellular compartments where diverse proteins involved in shared functions must efficiently assemble. Here, we demonstrate that proteins with shared functions share amino acid sequence codes that guide them to compartment destinations. A protein language model, ProtGPS, was developed that predicts with high performance the compartment localization of human proteins excluded from the training set. ProtGPS successfully guided generation of novel protein sequences that selectively assemble in targeted subcellular compartments. ProtGPS also identified pathological mutations that change this code and lead to altered subcellular localization of proteins. Our results indicate that protein sequences contain not only a folding code, but also a previously unrecognized code governing their distribution in specific cellular compartments.

biochemistry↗

bioRxiv2024

Immune regulators EDS1 and PAD4 constrain ADR1-dependent disease resistance in rice

Withdrawal StatementThe authors have withdrawn this manuscript owing to finding inconsistencies in genotyping of kitaake rice eds1 pad4 double mutant lines and to establishing that combined loss of EDS1 and PAD4 does not underlie the stunted autoimmune-like phenotype reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors: Jane Parker, Thomas Kroj and Haitao Cui (co-corresponding)

plant biology↗