Biology subjects

Lee, C.

Publications and source records attributed to Lee, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Longitudinal analysis of influenza A virus deletion-containing viral genomes reveals key determinants of co-evolutionary dynamics and interference

The substantial genetic diversity generated during influenza A virus replication facilitates both evasion of pre-existing host immunity and cross-species emergence. A major contributor to this diversity is the ubiquitous production of deletion-containing viral genomes (DelVGs) - viral RNAs with large internal deletions that arise during replication. DelVGs directly compete with wild-type (WT) genomes, and their accumulation has been implicated in modulating disease severity. However, the specific functional and genetic interactions between DelVGs and WT genomes remain poorly understood. To examine how DelVGs and WT genomes may co-evolve in mixed populations, we serially passaged influenza A virus under sustained high multiplicity-of-infection (MOI) conditions and used next-generation sequencing to build a longitudinal profile of DelVG emergence and dynamics. Early passages yielded a highly diverse repertoire of DelVGs across multiple segments, followed by a sharp contraction in overall diversity and the rise of one to two PB2-derived DelVGs that persisted at sustained high frequency. We identified a single-nucleotide substitution within PB2-derived DelVGs that significantly enhanced their replication and interference, whereas the same mutation proved lethal in the WT background. Collectively, these findings indicate that DelVGs are not mere byproducts of replication but dynamic genomic elements shaped by selection; their capacity to acquire adaptive mutations and outcompete WT genomes underscores their active role in shaping within-population viral dynamics.

microbiology↗

bioRxiv2025

Non-canonical DNA in bird telomere-to-telomere genomes

Non-canonical (non-B) DNA motifs are sequences that can fold into structures (e.g., G-quadruplexes and Z-DNA) distinct from the canonical right-handed helix. In mammals, these structures regulate gene expression, act as mutation hotspots, and are associated with cancer, yet they remain undercharacterized in other species. Because non-B DNA motifs are difficult to sequence, many are absent from incomplete genome assemblies, limiting functional analyses. Here, we present the first comprehensive analysis of non-B DNA motifs in birds, using the telomere-to-telomere genome of zebra finch, the near-complete chicken genome, and high-quality genomes of six additional bird species. We show that, first, unlike in mammals, the non-B DNA landscape in birds differs markedly among chromosome groups: gene-rich and extremely small dot chromosomes show the highest coverage (15.1-30.1% in zebra finch), microchromosomes--intermediate coverage (6.4-18.1%), and macrochromosomes--the lowest (5.9-6.9%). Non-B DNA motif coverage on dot chromosomes negatively correlates with PacBio sequencing depth, potentially explaining their assembly challenges. Second, similar to mammals, in zebra finch, G-quadruplexes are enriched at promoters and 5'UTRs, implying regulatory roles. We experimentally validated four common G-quadruplexes and predicted others using long-read methylation data. Overall, non-B DNA distribution reflects distinct features of avian genome architecture, suggests a role in gene regulation, and informs strategies for complete bird genome sequencing.

genomics↗

bioRxiv2025

Odyssey: reconstructing evolution through emergent consensus in the global proteome

We present Odyssey, a family of multimodal protein language models for sequence and structure generation, protein editing and design. We scale Odyssey to more than 102 billion parameters, trained over 1.1 x 1023 FLOPs. The Odyssey architecture uses context modalities, categorized as structural cues, semantic descriptions, and orthologous group metadata, and comprises two main components: a finite scalar quantizer for tokenizing continuous atomic coordinates, and a transformer stack for multimodal representation learning. Odyssey is trained via discrete diffusion, and characterizes the generative process as a time-dependent unmasking procedure. The finite scalar quantizer and transformer stack leverage the consensus mechanism, a replacement for attention that uses an iterative propagation scheme informed by local agreements between residues. Across various benchmarks, Odyssey achieves landmark performance for protein generation and protein structure discretization. Our empirical findings are supported by theoretical analysis.

synthetic biology↗

bioRxiv2025

The complete genome of a songbird

Bird genomes are the smallest among amniotes, but remain challenging to assemble due to their structural complexity. This study presents the first fully phased, diploid, telomere-to-telomere (T2T) reference genome for the zebra finch (Taeniopygia guttata), a model organism for neuroscience and evolutionary genomics. Combining multiple sequencing strategies resulted in closing nearly all gaps, adding [~]90 Mbp of previously missing sequence (7.8%). This includes T2T assemblies for all microchromosomes, including dot chromosomes, and the previously almost entirely missing chr16. The T2T genome is comprehensively annotated for genes, repeats, structural variants, and long-read methylation calls. Complete centromeric structures were assembled and annotated along with kinetochore binding sites. Relative to the previous high-quality reference of the Vertebrate Genomes Project, 2,778 (8.51%) previously unassembled or unannotated genes were identified, of which 9% overlap with segmental duplications. This first complete genome of a songbird, now the new public reference, illuminates avian genome architecture and function.

genomics↗

bioRxiv2025

Dual-patterned pluripotent stem cells self-organize into a human embryo model with extended anterior-posterior patterning

Human gastruloids are a powerful class of stem cell-derived models that recapitulate key features of early embryonic development, including symmetry breaking and the emergence of three germ layers1-3. However, they lack anterior embryonic structures and coordinated axial organization4-6. To address this limitation, we pre-patterned human pluripotent stem cells (hPSCs) by exposing them to either anterior (FGF2) or posterior (CHIR99021 [CHIR] & retinoic acid [RA]) cues. Upon mixing, these dual-patterned hPSCs interacted and self-organized into elongated structures with both anterior and posterior features--which we term anterior-posterior (AP) human gastruloids. Anteriorly pre-treated cells robustly intercalated into posteriorly pre-treated cells, collectively giving rise to a continuum of neural tissues--including a brain-like domain, a neural tube-like structure, and neuro-mesodermal progenitors (NMPs)--with segmented somites arrayed bilaterally. Single cell RNA sequencing (scRNA-seq) revealed that human AP gastruloids contain cell types resembling the midbrain-hindbrain boundary (MHB), regionalized hindbrain structures (i.e. rhombomeres 1-8), regionalized neural crest (i.e. cranial, vagal, trunk)7,8 and head mesoderm. Transcriptomic comparisons to primate embryos revealed that human AP gastruloids most closely resemble Carnegie stage 11 (CS11) embryos. While they lack a notochord and full dorsal-ventral polarity, human AP gastruloids recapitulate key spatial and temporal features of early neurulation and somitogenesis. Perturbation of folic acid metabolism or rho-associated kinase (ROCK) signaling induced spinal cord defects, phenocopying aspects of spina bifida and other neural tube defects, highlighting this models potential for studying congenital disorders9. AP gastruloids may serve as a simple, robust, scalable platform for modeling coordinated human AP body axis development. More broadly, our results suggest that controlled interactions between differentially prepatterned progenitors can initiate self-organization of complex body axis features. The "pattern-and-mix" strategy may serve as a generalizable framework for assembling spatially organized stem cell models of mammalian development.

developmental biology↗

bioRxiv2025

VSA-2, a novel plant-derived adjuvant for SARS-CoV-2 subunit vaccine

QS-21, a key component of several licensed vaccines is facing limited supply, dose-limiting toxicity and other drawbacks which together limit its broader usage. Development of saponin alternatives to QS-21 that retain its desirable adjuvant activity without its drawbacks is in high need. Incorporating an amide side chain into the more sustainable Momordica saponins (MS) I and II led to the recent discovery of two semisynthetic immunostimulatory adjuvants VSA-1 and VSA-2. Here, we showed that the receptor-binding protein (RBD) of ancestral SARS-CoV-2 adjuvanted with VSA-2 (VSA-2-RBD) induced high titers of SARS-CoV-2-specific humoral and T helper-1 prone immune responses in mice comparable to that triggered by QS-21-RBD. Vaccination with VSA-2-RBD provided strong protection against SARS-COV-2, Delta and Omicron variants infection and the virus-induced lung inflammation and pathology similarly as QS-21-RBD vaccination. Overall, our results suggest that VSA-2 adjuvant can potentially complement the clinically proven saponin adjuvant QS-21 in vaccines against infectious diseases.

microbiology↗

bioRxiv2025

PEDOT:PSS conducting eutectogel for enhanced electrical recording and stimulation in implantable neural interfaces

Conductive polymers such as PEDOT:PSS are widely used in bioelectronic interfaces due to their mixed ionic-electronic conductivity and biocompatibility. However, their mechanical fragility and limited processability constrain their performance in implantable devices. Deep eutectic solvents (DES), when combined with PEDOT:PSS, form eutectogels that enable thick, soft coatings. Here, we present a PEDOT:PSS-based eutectogel incorporating choline chloride: lactic acid and GOPS, integrated into flexible thin-film electrode arrays for sciatic nerve interfacing. These implants feature an array of electrodes and a pre-formed spiral geometry to conformally wrap small-diameter nerves. Devices were fabricated using standard photolithography and reactive ion etching techniques, allowing side-by-side comparison of PEDOT:PSS/DES with conventional PEDOT:PSS electrodes. PEDOT:PSS/DES enabled single-layer films up to 800 nm thick, significantly greater than PEDOT:PSS, and yielding over two-fold improvements in impedance and charge injection capacity in vitro. Acute in vivo electrophysiology in rats confirmed enhanced neural recording and stimulation capabilities, with lower impedance, higher capacitance, and reduced motor activation thresholds. While PEDOT:PSS/DES more reliably elicited motor responses at lower stimulation currents, electromyogram signal amplitudes from the tibialis anterioris at matched stimulation levels were comparable between materials. These results suggest that while superior electrochemical properties improve neural interface performance, local electrode-tissue interactions remain critical. Overall, this work establishes DES-modified PEDOT:PSS as a promising electrode material for soft neural interfaces and highlights its potential for advancing implantable bioelectronics.

neuroscience↗

bioRxiv2025

High-stability, clamp-free soluble Sarbecovirus spike trimers and their potential for pan-Sarbecovirus vaccine development.

Broadly effective vaccines are needed to protect against future pandemics caused by severe acute respiratory syndrome coronavirus (SARS CoV)-like coronaviruses (sarbecoviruses). The development of simple trimeric subunit vaccines based on the Sarbecovirus spike (S) has proven problematic due to the unstable nature of the S trimer. Here we developed clamp-free, highly stable soluble S trimers by truncating the stem helix to maximize yield and covalently linking the 3 monomers via engineered disulfides to increase thermostability. In K18hACE2 mice, covalently linked SARS CoV-2 S trimers elicited >10-fold higher neutralizing antibody (NAb) titres than parental unlinked trimers and protected the mice against viral challenge. A trivalent vaccine formulation comprised of covalently stabilized spikes derived from 3 divergent ACE2-using Sarbecovirus clades elicited broad and potent neutralizing activity in mice. The covalently linked S trimers were stable at 37{degrees}C for 112 days and remained intact following lyophilization and storage at ambient temperature for 6 months. This study establishes a framework for producing simple and stable highly immunogenic pan-Sarbecovirus S subunit vaccines that can be stored and distributed in the absence of a cold chain.

microbiology↗

bioRxiv2025

Structural Variants at Intronic CTCF Loop Anchors Drive Differential Exon Usage

CTCF-mediated chromatin loops are known to influence gene regulation, yet their role in pre-mRNA splicing remains incompletely understood. Here, we demonstrate that structural variants (SVs) at the anchors of intronic CTCF loops can modulate exon usage. By integrating high-resolution three-dimensional (3D) genome organization and gene expression datasets from C57BL/6J (B6) and 129S1/SvImJ (129S) mouse embryonic stem cells (ESCs), with structural variant (SV) maps from the 129S mouse, we identified thousands of intron-anchored CTCF loops. Our data indicate that SVs intersecting loop anchors are more frequently associated with differential exon inclusion events than with changes in overall gene expression. CRISPR/Cas9 deletion of two SV-harboring intronic CTCF sites in Numbl and Ireb2 validated the predicted splicing shifts observed between B6 and 129S ESC that correspond with diminished long-range chromatin looping. Our findings reveal a direct mechanistic link between 3D genome architecture and alternative splicing and highlight non-coding SVs as modulators of transcript diversity. Our study has thus identified a novel class of CTCF-bound regulatory elements regulating alternative splicing. Cataloging and validating these functional elements will elucidate molecular mechanisms underlying phenotypic variation within populations.

molecular biology↗

bioRxiv2025

Extreme somatic mutation variation through time and space in walnut clones

Many plants, unlike most animals, can reproduce as clones1. Cloning plants is critical for agriculture2,3 and biotechnology4, but the extent of somatic mutation arising during different propagation methods remains an important question for both fundamental research and agriculture. Here, we discover a surprisingly complex mutational history in a multi-decade natural experiment of isogenic walnut clones derived and maintained through three alternative methods: budwood propagation of field-grown trees, in vitro shoot culturing, and in vitro somatic embryogenesis. We generated a haplotype-phased reference genome assembly and revealed a >3500% increase in somatic embryo mutation rates compared to field-grown trees, along with a distinct mutation spectrum. The assembly also helped reveal extreme genomic instability in the somatic embryos, including multiple chromosomal duplications, megabase-scale deletions, telomere expansions, somatic recombination events, and ongoing transposable element activation. Our survey of somatic mutation also provides high-resolution insight into clonal stem cell dynamics, confirming the canonical meristem layers of flowering plants in the tree and shoot clones, while uncovering clear evidence of frequent single-cell bottlenecks in the somatic embryos. These discoveries inform practical questions about mutagenesis through plant tissue culture and serve as a benchmark to complement emerging paradigms of somatic mutation research in humans and other organisms.

genomics↗

bioRxiv2025

Ras-Responsive Element Binding Protein 1 regulates survival of Group 3 medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and Group 3 (G3, MYC-driven) MB has the worst prognosis. Despite advances in molecular classification, oncogenic drivers of G3 MB remain poorly defined. To identify such drivers, we profiled transcription factor expression across MB subgroups. Our analysis revealed Ras-responsive element binding protein 1 (RREB1) as one of the most highly expressed transcription factors in G3 MB. RREB1 knockdown impaired cell proliferation in vitro and prolonged survival in orthotopic xenograft models, suggesting it plays a key role in regulating tumor growth. Mechanistically, RREB1 acts by enhancing transcription of TGF-{beta} pathway genes. Upstream, RREB1 expression is controlled by c-MET signaling, and the MET inhibitor SU11274 decreased RREB1 levels and MB cell viability. Local delivery of SU11274 in tumor-bearing mice suppressed RREB1 expression and extended survival. These results establish the c-MET/RREB1 axis as a critical oncogenic regulator and a promising therapeutic target in in high-risk MB.

cancer biology↗

bioRxiv2025

Genomic and morphometric evidence for Austronesian-mediated pig translocation in the Pacific

Human mediated translocation of non-native pig species (genus Sus) to the islands of Wallacea and Oceania has significantly altered local ecosystems. To investigate the timing and trajectory of these introductions, we conducted both genomic analyses of 576 pig nuclear genomes and a geometric morphometric analysis of 714 modern and ancient dental remains. Our analyses demonstrate that feral and domestic pigs in Wallacea and Oceania possess diverse ancestries resulting from the introduction of multiple, sequential pig populations followed by gene flow. Despite the variability in their genomic ancestry these pigs all possess a distinct tooth morphology, and a genetic link to the Chinese domestic pig populations that accompanied the dispersal of Austronesian language speakers [~]4,000-3,000 years ago via Taiwan and the Philippines.

evolutionary biology↗

bioRxiv2025

Physical and functional interaction of Lrrc56 and Odad3 controls deployment of axonemal dyneins in vertebrate multiciliated cells

Primary ciliary dyskinesia is a genetically heterogeneous motile ciliopathy characterized chronic respiratory disease, laterality defects, hydrocephalus, and infertility, caused by impaired function of motile cilia. LRRC56 has recently emerged as a novel PCD candidate gene, but its role in vertebrate cilia remains poorly understood. Here, we used Xenopus laevis multiciliated cells, targeted knockdown, and in vivo imaging to investigate Lrrc56 function. We show that loss of lrrc56 causes specific depletion of outer dynein arms (ODAs) from the distal axoneme. In vivo affinity purification mass spectrometry revealed that Lrrc56 binds the ODA docking complex components, including Odad3. Consistently, Lrrc56 knockdown also led to distal loss of Odad3. Moreover, we show that disease-associated variants in LRRC56 and ODAD3 disrupted their localization and interaction, pointing to a shared functional pathway. Our work demonstrates that Lrrc56 is a critical regulator of distal ODAs and ODA docking complex deployment and provides new mechanistic insight into how LRRC56 mutations contribute to PCD.

cell biology↗

bioRxiv2025

SpatioEv: Spatial evolution of protein and morphological features reveals development dynamics of cells and spatial neighbourhoods

Understanding cellular function in tissues demands sophisticated tools to decode complex microenvironmental interactions. Current spatial analysis methods often lack the comprehensive framework needed to systematically analyse cell morphology, dynamics, interactions, and extracellular matrix (ECM) architecture. We introduce SpatioEv, a unified computational framework for highly multiplexed tissue imaging that addresses these critical gaps. SpatioEv integrates automated quality control, cell phenotyping, neighbourhood identification, multi-scale spatial characterization, niche boundary analysis, ECM fiber-cell interaction mapping, and spatiotemporal trajectory inference. This pipeline enables reproducible cell annotation, reveals novel ECM-cell interactions, characterizes tissue neighbourhood boundaries, and infers developmental progressions directly from spatial data. Using this, we can identify disease-specific spatial signatures distinguishing rheumatoid arthritis from osteoarthritis, characterize diverse tumour boundary phenotypes in cancer metastases in liver, and map evolutionary trajectories in pancreatic ductal adenocarcinoma (PDAC) at single-niche resolution. Our findings highlight the significance of spatial context in shaping cell behaviour and underscore its potential to uncover emergent tissue architecture and cellular dynamics. By addressing major analytical challenges, SpatioEv provides a scalable, adaptable platform for advancing spatial biology and translational research.

systems biology↗

bioRxiv2025

Cell layer-specific modification of cell wall is associated with exo-mesocarp split in pistachio (Pistacia vera L.)

Pistachio (Pistacia vera) is a drought and salinity-tolerant perennial whose fruit features a fleshy exo-mesocarp, or "hull," that protects the kernel. Hull development and degradation are key to kernel quality, yet the anatomy and mechanisms driving hull breakdown during late-stage development remain largely unknown. Here, we show that the hull contains anatomically distinct layers of hypodermal parenchyma and filler parenchyma. Using a combination of transcriptome analyses and immunohistochemistry, we show that changes in pectin associated gene expression and modification of this polysaccharide are involved in hull cell size increase, loss of cell-cell adhesion, and softening. Anatomical analysis shows that filler parenchyma expands during late-stage hull development while hypodermal parenchyma remains constant in size. Field data suggest that irrigation and humidity affect pistachio hull split, implicating a role for water status in cell expansion. In summary, the complex interplay between molecular, cellular, and environmental changes suggests that cell layer-specific modifications of the cell wall are linked to exo-mesocarp splitting, forming a model for understanding the mechanism of fruit split during ripening in non-berry fruit crops. HighlightCell-layer specific modifications of the cell wall are associated with cell expansion and loss of cell-cell adhesion, leading to hull split during late-stage pistachio fruit development.

plant biology↗

bioRxiv2025

Transposon-plasmid nesting enables fast response to fluctuating environments

Mobile genetic elements (MGEs) play a critical role in shaping the response and evolution of microbial populations and communities. Despite distinct maintenance mechanisms, different types of MGEs can form nested structures. Using bioinformatics analysis of 14,338 plasmids in the NCBI RefSeq database, we found transposons to be widespread and significantly enriched on plasmids relative to chromosomes, highlighting the prevalence of transposon-plasmid nesting. We hypothesized that this nested structure provides unique adaptive advantages by combining transposition-driven genetic mobility with plasmid-mediated copy number amplification. Using engineered transposon systems, we demonstrated that nesting enables rapid and tunable responses of transposon-encoded genes in fluctuating environments. Specifically, transposition maintains a reservoir of the encoded genes, while plasmid copy number fluctuations further amplify the dynamic range of gene dosage, thus enhancing the response speed and stability of transposon-encoded traits. Our findings demonstrate an adaptive benefit of transposon-plasmid nesting and provide insights into their ecological persistence and evolutionary success.

systems biology↗

bioRxiv2025

The Rationalization of Carbon Monoxide and Hemoglobin Association

Carbon monoxide is a colorless, odorless, and poisonous gas, responsible for approximately 100,000 emergency room visits and over 420 deaths in the U.S. each year. Both carbon monoxide and oxygen bind to the ferrous ions in hemoglobin, but carbon monoxide has a significantly higher affinity. Extensive research has been conducted on the interaction between carbon monoxide and hemoglobin. However, a straightforward and practically applicable equation describing the relationship between carbon monoxide saturation and pressure is not found in the existing literature. In this paper, we establish an equation and confirm that the plot of CO saturation against CO pressure follows a hyperbolic shape, characterized by a continuous decrease in slope. In contrast, the oxygen-hemoglobin association curve is sigmoidal. These distinct curve shapes have different physiological implications. Our equation enables the determination of one variable--either saturation or pressure--if the other is known. Further analysis reveals the distribution of all five species of carboxyhemoglobin, showing that the triply bound form is abundant--a notable contrast to the distribution of oxyhemoglobin species. Additionally, our equation confirms that carbon monoxides affinity for hemoglobin is approximately 230 times higher than that of oxygen. Lastly, we propose a new general equation that may generate all carbon monoxide-hemoglobin association curves under various oxygen pressures. New and NoteworthyIn this paper, we introduce the first known set of equations that model the Carbon Monoxide Hemoglobin association curve and the Carbon Monoxide Hemoglobin Fractional association curves. These novel equations represent a noteworthy advancement in the ability to quantitatively model CO binding to hemoglobin. We present the methodology utilized that explains the derivation of precise coefficients for these equations, along with the data points used to calculate the coefficients. Unlike existing models, our equations allow the direct computation of the CO saturation from the CO association equation and the CO fraction association equations from the partial pressure of carbon monoxide. This approach simplifies the mathematical modeling process while preserving a high degree of biological significance. Furthermore, the equations are built upon a complex and precise methodological foundation, enabling a deeper understanding of the binding behavior of hemoglobin in response to varying CO levels. This quantitative model has the potential to be applied to clinical practices in medicine. It provides insight into the complex relationship between CO and hemoglobin that is critical to many biological processes.

physiology↗

bioRxiv2025

IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

IFN-{gamma} is secreted by multiple lymphoid subsets in response to antigen stimulation and can reprogram and prime macrophages epigenetically and transcriptionally to increase responses to inflammatory stimuli such as LPS, IL-1{beta} or TNF-. IFN-{gamma}-driven M1-like inflammatory macrophage states are shared across human immune-mediated inflammatory diseases (IMIDs) and while IFN-{gamma} is nonredundant for defense to intracellular pathogens it is unclear if this is also the case in IMIDs. To identify additional secreted ligands which could prime and induce M1-like macrophages we screened >600 human proteins in human primary macrophages. Using complementary functional genomics approaches, we discovered that IL-32{beta} induced an M1-like inflammatory state in non-primed and IFN-{gamma}-primed macrophages. IL-32{beta} induced signaling, transcriptional, tolerance, cross-tolerance, and inflammatory responses in macrophages which were MyD88, IRAK1 and Myddosome-dependent. These responses to host IL-32{beta} were similar to yet distinct from, those induced by microbial LPS. IL-32 protein was elevated in serum from patients with severe COVID-19 and IL-32{beta} together with IFN-{gamma} were expressed by T cells and induced a macrophage transcriptional response which was shared by monocytes and macrophages in mild and severe COVID-19.

immunology↗