Biology subjects

Chen, Y.-C.

Publications and source records attributed to Chen, Y.-C..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Mineralized Tissue-Targeting Expression System for Local Control of Gene Expression

Targeted control of gene expression in mineralized tissue would enable the use of nucleic acids to modulate the local microenvironment at diseased sites, ultimately promoting bone regeneration. Piperazine-linked bisphosphonate ionizable lipids provide a facile approach to targeting the transfection of mineralized tissue with lipid nanoparticles (LNPs). Here, we develop a Mineralized Tissue-Targeting Expression System (MiTEX) using bisphosphonate LNPs to locally target mineralized tissues by adsorption to mineral surfaces and bone graft materials. MiTEX demonstrated a significant increase in the adsorption of RNA onto hydroxyapatite substrates, which retained the ability to transfect bone mesenchymal cells via the adsorbed layer of mRNA LNPs. Bone graft scaffolds functionalized by adsorbed Cre mRNA-LNP were implanted to genetically label newly formed bone tissues in vivo. The surface affinity and adsorption of bisphosphonate lipids provided a local reservoir in mineralized tissues, sustaining the in vivo delivery of MiTEX. Furthermore, the targeted delivery of RNA therapeutics was demonstrated using STAT3 siRNA to modulate gene expression and proinflammatory cytokine release in ex vivo periodontal tissues. The design of this new RNA-functionalized delivery platform will promote the development of precision nucleic acid therapeutics for local anti-inflammatory treatments and bone regeneration at mineralized tissue interfaces.

bioengineering↗

bioRxiv2025

Cytoskeleton remodeling caused by keratin dysregulation triggers tumor aggressiveness via promoting genomic instability and cellular adaption

Cellular architecture depends on keratin intermediate filament as a fundamental component, which offers essential mechanical support to fight environmental stresses. Our previous research demonstrated that keratin fusion variants increase tumor aggressiveness through enhanced cancer stemness in oral squamous cell carcinoma. The proper functioning of keratins plays an essential role in maintaining cell structure while determining cell fate. The present study demonstrates that keratin fusion variant drives genomic instability through cytokinesis defects, which results in the formation of polyaneuploid cancer cells (PACC). The cells expressing keratin fusion show elevated DNA damage repair gene expression, which serves as a key factor for mitotic slippage during cancer development. The PACCs generated by keratin fusion make cancer cells resistant to cisplatin treatment while simultaneously reducing {gamma}-H2AX induction and increasing survival rates. The Gene Set Enrichment Analysis results showed increased "regulation of actin cytoskeleton" activity in keratin fusion-expressing cells correlated with elevated actin filament networks and increased cell motility in these cells. In summary, the keratin fusion variant enhances cancer aggressiveness through three mechanisms: it creates genomic instability that leads to PACC formation and enables cancer cells to evade cGAS/STING-mediated death signals and modifies cytoskeleton structures, which results in drug resistance and metastasis.

cancer biology↗

bioRxiv2025

Structural Elucidation and Revised Biosynthetic Pathway of the Membrane Vesicle-Associated Antifungal Compound AFC

Bacterial membrane vesicles (MVs) serve as delivery vehicles for hydrophobic and membrane-associated secondary metabolites, enhancing their solubility, stability, and bioactivity. Here, we show that the antifungal compound AFC-BC11 (AFC), produced by Burkholderia cenocepacia K56-2, is selectively packaged into and released via MVs. Using HR-MS/MS, NMR, and stable isotope feeding experiments, we determined the chemical structure of AFC and analyzed its biosynthesis. Our results confirm that the structure largely matches the recent report by Zhong et al., with a key difference: the double bond in the fatty acid moiety is positioned between C11 and C12. We provide compelling evidence that this constitution reflects the direct incorporation of cis-vaccenic acid, the most abundant fatty acid in B. cenocepacia, rather than a tailoring modification. Comparative analysis of afcU, afcF, and afcS mutants suggests a biosynthetic pathway involving {omega}-modification of cis-vaccenic acid, revising previous proposals of citric acid conjugation to myristic acid and opening avenues for acyl chain engineering. Together, these findings establish AFC as an MV-associated antifungal metabolite, provide a refined structural and biosynthetic model, and highlight the role of MVs in the dispersal of hydrophobic bioactive compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/685075v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f4d736org.highwire.dtl.DTLVardef@15d2683org.highwire.dtl.DTLVardef@8b891corg.highwire.dtl.DTLVardef@d6b693_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Matrix Stiffness Governs Fibroblast-Driven Immune Homeostasis in Gingival Tissues

Periodontal disease is associated with inflamed gingival tissues and degradation of the gingival extracellular matrix (ECM), yet the role of mechanical cues is poorly understood. Gingival ECM in periodontal disease showed a loss of fibrillar collagen compared to healthy samples. We hypothesized that ECM softening in periodontal disease contributes to inflammation due to dysregulated gingival fibroblasts (GFs). A mechanically tunable hydrogel model of the gingival ECM was developed to investigate the mechano-immune crosstalk. Stiff collagen-alginate hydrogels matched the rheological properties of gingival biopsies. Human donor GFs encapsulated in these stiff hydrogels showed significantly suppressed toll-like receptor inflammatory responses compared to soft. Stiffness-dependent inflammatory responses of GFs were directed by the non-canonical NF{kappa}B pathway and epigenetic nuclear organization. The direct impact of mechanical cues on immune responses was investigated with human donor cells ex vivo by co-culture of human GFs with myeloid cells and in human gingival explants. Myeloid progenitors co-cultured with GFs in stiff hydrogels differentiated into immunomodulatory dendritic cells. Ex vivo crosslinking of human gingival tissue increased stiffness and reduced inflammatory cytokines. Gingival mechano-immune regulation provides a new avenue for biomaterials-based treatments in periodontitis.

bioengineering↗

bioRxiv2025

Linked selection of insertions and deletions in coding regions of the great tit genome

Insertions and deletions (indels) are a ubiquitous form of genomic variation; yet, their contribution to adaptive evolution remains poorly understood. Technical challenges in identifying indels and integrating them into existing evolutionary models have limited their study, but previous work suggests that they may be subject to strong selective pressures. Here, we investigated selective signatures of indels in coding regions of the great tit (Parus major). We identified species-specific indels in protein-coding genes and analyzed the 300 bp flanking regions of indels using codeml to estimate dN, dS, and{omega} (dN/dS). Additionally, we performed enrichment analyses and protein structure modeling to assess functional consequences. We found that mutations in the flanking regions of indels are non-random and that selection strength varies with indel type and length. Genes including indels were enriched for functions related to protein and nucleic acid binding and linked to potentially adaptive traits such as vocal learning, migration, circadian rhythm, and environmental acclimation. Our results suggest that insertions and deletions may serve as hotspots of selection, influencing nearby sequences and contributing to adaptation in the great tit. Significance StatementSmall DNA changes called insertions and deletions (indels) are common in genomes, but their role in evolution is often overlooked because they are difficult to study. Using the genome of the great tit, a well-studied songbird, we identified thousands of indels in protein-coding regions and examined how they affect nearby DNA. We found that indels are not neutral accidents: they show signs of natural selection and may influence important traits. Some of the affected genes are linked to behaviors and adaptations that are crucial for survival, such as vocal learning, migration, and adaptation to new environments. These results suggest that indels can act as hotspots for evolutionary change and may help explain how species adapt to their surroundings. By high-lighting the evolutionary importance of these often-neglected mutations, our study opens new perspectives on the genetic basis of adaptation.

evolutionary biology↗

bioRxiv2025

Discovery and performance of DNA methylation panels for cancer detection and classification in blood

Examining DNA in a liquid biopsy for non-invasive cancer detection relies on identifying dilute signal in a high background. This study aims to identify DNA methylation biomarkers for multi-cancer detection. Utilizing large tissue datasets, we apply novel search algorithms to discover confined biomarker panels capable of distinguishing tumor from normal and determining the tissue of origin. We explore the applicability to blood-based testing using targeted methylation sequencing followed by machine learning classification. We present an 8-marker panel, which successfully predicts tumors across 14 types with a 91% average sensitivity, maintaining a low false positive rate (< 0.04%). Additionally, a panel of 39 CpG sites exhibits accuracies ranging from 69% to 98% for identifying tissue of origin. When tested on 114 patient plasma samples (colon, liver, pancreatic, prostate, and stomach cancer), the 8-marker panel obtains an AUC of 0.78 with a 78% sensitivity among 32 early-stage patients (stage I-II), and 60% overall. Using the 39-marker panel in a multi-class classification model selecting only the best match, 54% of tumor samples were on average correctly assigned to the tissue of origin, and up to 80% when allowing more inclusive criteria. Using a limited set of biomarkers, our work contributes to advancing non-invasive cancer diagnostics.

cancer biology↗

bioRxiv2025

Regulatory logic of neuronal identity specification in Drosophila

Combinations of terminal selector transcription factors (tsTFs) are thought to establish and maintain the unique identities of the numerous cell types found in nervous systems. However, it remains largely unclear how tsTF combinations are specified during development, and how they then coordinate the type-specific differentiation programs of each neuron. To investigate these regulatory mechanisms, we performed simultaneous single-cell RNA and ATAC sequencing on the Drosophila optic lobes at four stages of their development and identified over 250 distinct cell types. We characterized the common cis-regulatory features of neuronal enhancers and performed comprehensive inference of gene regulatory networks across cell types and stages. Our results reveal cell-type and stage-specific enhancers of many neuronal genes and the cooperative actions of tsTFs, pan-neuronal and ecdysone-responsive TFs on these enhancers. We show that the same effector genes are often regulated by different tsTF combinations acting through distinct enhancers in different neurons. During neurogenesis, tsTF codes are established within a brief critical period in newborn neurons, often through cell-type-specific enhancers that are not accessible in their progenitors. Accordingly, when neuroblast temporal patterning TFs are re-utilized as tsTFs in neurons, they are regulated independently through separate enhancers. Therefore, neuronal identity specification and differentiation is a multi-step regulatory program, wherein the same TFs enact distinct regulatory codes at different steps and across cell types.

neuroscience↗

bioRxiv2025

SwarmGenomics: A Unified Pipeline for Individual-Based Whole-Genome Analyses

Advances in sequencing technologies have made whole-genome data widely accessible, enabling research in population genetics, evolutionary biology, and conservation. However, analyzing whole-genome sequencing (WGS) data remains challenging, often requiring multiple specialized tools and substantial bioinformatics expertise. We present SwarmGenomics, a modular, user-friendly command-line pipeline for reference-based genome assembly and individual-based genetic analyses. The pipeline integrates seven modules: heterozygosity estimation, runs of homozygosity detection, Pairwise Sequentially Markovian Coalescent (PSMC) analysis, unmapped reads classification, repeat analysis, mitochondrial genome assembly, and nuclear mitochondrial DNA segment (NUMT) identification. Each module can be run independently or as part of a complete workflow. We demonstrate the pipelines utility with a case study on the giant panda (Ailuropoda melanoleuca), revealing insights into genetic diversity, inbreeding history, historical population size changes, transposable element activity, and microbial contamination. SwarmGenomics lowers the entry barrier for genomic analysis of diploid, non-model species, serving both as a research and teaching tool. The pipeline and documentation are available at https://github.com/AureKylmanen/Swarmgenomics.

genomics↗

bioRxiv2025

Title: Retinal Calcium Waves Coordinate Uniform Tissue Patterning of the Drosophila Eye.

Optimal neural processing relies on precise tissue patterning across diverse cell types. Here, we show that spontaneous calcium waves arise among non-neuronal support cells in the developing Drosophila eye to drive retinal morphogenesis. These waves are initiated by Cad96Ca receptor tyrosine kinase signaling, triggering PLC{gamma}-mediated calcium release from the endoplasmic reticulum. A cell-type-specific Innexin code coordinates wave propagation through a defined gap junction network among non-neuronal retinal cells, excluding photoreceptors. Wave intensity scales with ommatidial size, triggering stronger Myosin II-driven apical contraction at interommatidial boundaries in larger ommatidia. This size-dependent mechanism compensates for early boundary irregularities, ensuring uniform ommatidial packing critical for precise optical architecture. Our findings reveal how synchronized calcium signaling among non-neuronal cells orchestrates tissue patterning in the developing nervous system.

developmental biology↗

bioRxiv2025

High-resolution comparative single-cell transcriptomics of doublesex-expressing neurons reveals evolutionary conservation and diversity of sexual circuits in Drosophila

Understanding how the cellular and molecular composition of neural circuits change during evolution is essential for deciphering how behavior evolves. Male sexual behaviors in Drosophila species are remarkably diverse, and the underlying sexual circuits are specified by sex determination genes. Here, we employed single-cell transcriptomics to systematically characterize and compare neuron cell types that express the sex determination gene doublesex in adult males across Drosophila species with divergent sexual behaviors. High-resolution profiling to delineate cellular diversity revealed a largely conserved set of cell types across four species, with minimal evolutionary gain or loss and potentially more prevalent changes in cell type abundance. In-depth comparisons between D. melanogaster and D. yakuba showed that transcriptomic conservation and differentially expressed genes between species are highly cell-type-specific, suggesting that cell types evolve as highly independent units. We identified widespread species differences in gene expression, particularly in the neuromodulatory signaling pathway, while preserving a conserved circuit layout in sexual identity and neurotransmitter properties. We further generated a female dataset in D. melanogaster to define sex differences in cell types and examine species divergence in relation to sex differences. Finally, we reported marker gene combinations that uniquely define each cell type, providing a foundational resource that enables the design of cell -type-specific genetic reagents. Overall, our study provides fundamental insights into the cellular diversity of sexual circuits and how evolution shapes cell types and gene expression in behavioral adaptations.

evolutionary biology↗

bioRxiv2025

Pluripotent Stem Cell Plasticity is Sculpted by a Slit-Independent Robo Pathway in a Regenerative Animal

Whole-body regeneration requires adult stem cells with high plasticity to differentiate into missing cell types. Planarians possess a unique configuration of organs embedded in a vast pool of pluripotent stem cells. How stem cells integrate positional information with discrete fates remains unknown. Here, we use the planarian pharynx to define the cell fates that depend on the pioneer transcription factor FoxA. We find that Roundabout receptor RoboA suppresses aberrant pharynx cell fates by altering foxA expression, independent of the canonical ligand Slit. An RNAi screen for extracellular proteins identifies Anosmin-1 as a potential partner of RoboA. Perturbing global patterning demonstrates that roboA/anosmin-1 functions locally in the brain. By contrast, altering pharynx fate with foxA knockdown induces head-specific neurons in the pharynx, indicating a latent plasticity of stem cells. Our data links critical extracellular cues with cell fate decisions of highly plastic stem cells, ensuring the fidelity of organ regeneration.

developmental biology↗

bioRxiv2025

Temporal and Notch identity determine layer targeting and synapse location of medulla neurons

How specification mechanisms that generate neural diversity translate into specific neuronal targeting, connectivity, and function in the adult brain is not understood. In the medulla region of the Drosophila optic lobe, neural progenitors generate different neurons in a fixed order by sequentially expressing a series of temporal transcription factors as they age. Then, Notch signaling in intermediate progenitors further diversifies neuronal progeny. Using the Electron Microscopy reconstruction of the adult fly brain, we matched the temporal and Notch identity of medulla neurons with their adult targeting features across the different optic lobe neuropils at synapse resolution, and found that these specification mechanisms are significantly associated with the depth of neuropil targeting in the adult brain, for both local interneurons and projection neurons. We show that this temporal identity-dependent targeting of projection neurons unfolds early in development and is genetically determined. Notably, synapse location in the adult brain of putative medulla neurons can be used to predict their temporal origin. Moreover, synapses of neuronal types from different connectome-predicted functional subsystems are compartmentalized in the different neuropils, suggesting a link between synaptic stratification and specific visual functions. Finally, we show that candidate terminal selector TFs are significantly associated with a specific depth bias in the optic lobe neuropils, providing putative molecular determinants of this targeting. Together, we show that temporal identity and Notch status of medulla neurons can predict their neuropil synapse location, linking their developmental patterning with adult circuit architecture.

developmental biology↗

bioRxiv2024

An Interbacterial Cysteine Protease Toxin Inhibits Cell Growth by Targeting Type II DNA Topoisomerases GyrB and ParE

Bacteria deploy a diverse arsenal of toxic effectors to antagonize competitors, profoundly influencing the composition of microbial communities. Previous studies have identified an interbacterial toxin predicted to exhibit proteolytic activity that is broadly distributed among Gram-negative bacteria. However, the precise mechanism of intoxication remains unresolved. Here, we demonstrate that one such protease toxin from Escherichia coli, Cpe1, disrupts DNA replication and chromosome segregation by cleaving conserved sequences within the ATPase domain of type II DNA topoisomerases GyrB and ParE. This cleavage effectively inhibits topoisomerase-mediated relaxation of supercoiled DNA, resulting in impaired bacterial growth. Cpe1 belongs to the papain-like cysteine protease family and is associated with toxin delivery pathways, including the type VI secretion system and contact-dependent growth inhibition. The structure of Cpe1 in complex with its immunity protein reveals a neutralization mechanism involving competitive substrate binding rather than active site occlusion, distinguishing it from previously characterized effector-immunity pairs. Our findings unveil a unique mode of interbacterial intoxication and provide insights into how bacteria protect themselves from self-poisoning by protease toxins.

microbiology↗

bioRxiv2024

Detecting Clinically Relevant Topological Structures in Multiplexed Spatial Proteomics Imaging Using TopKAT

Novel multiplexed spatial proteomics imaging platforms expose the spatial architecture of cells in the tumor microenvironment (TME). The diverse cell population in the TME, including its spatial context, has been shown to have important clinical implications, correlating with disease prognosis and treatment response. The accelerating implementation of spatial proteomic technologies motivates new statistical models to test if cell-level images associate with patient-level endpoints. Few existing methods can robustly characterize the geometry of the spatial arrangement of cells and also yield both a valid and powerful test for association with patient-level outcomes. We propose a topology-based approach that combines persistent homology with kernel testing to determine if topological structures created by cells predict continuous, binary, or survival clinical endpoints. We term our method TopKAT (Topological Kernel Association Test) and show that it can be more powerful than statistical tests grounded in the spatial point process model, particularly when cells arise along the boundary of a ring. We demonstrate the properties of TopKAT through simulation studies and apply it to two studies of triple negative breast cancer where we show that TopKAT recovers clinically relevant topological structures in the spatial distribution of immune and tumor cells.

bioinformatics↗

bioRxiv2024

Mu-opioid receptor activation potentiates excitatory transmission at the habenulo-peduncular synapse

The continuing opioid epidemic poses a huge burden on public health. Identifying the neurocircuitry involved and how opioids modulate their signaling is essential for developing new therapeutic strategies. The medial habenula (MHb) is a small epithalamic structure that projects predominantly to the interpeduncular nucleus (IPN) and represents a mu-opioid receptor (MOR) hotspot. This habenulo-peduncular (HP) circuit can regulate nicotine and opioid withdrawal; however, little is known about the physiological impact of MOR on its function. Using MOR-reporter mice, we observed that MORs are expressed in a subset of MHb and IPN cells. Patch-clamp recordings revealed that MOR activation inhibited action potential firing in MOR+ MHb neurons and induced an inhibitory outward current in IPN neurons, consistent with canonical inhibitory effects of MOR. We next used optogenetics to stimulate MOR+ MHb axons to investigate the effects of MOR activation on excitatory transmission at the HP synapse. In contrast to its inhibitory effects elsewhere, MOR activation significantly potentiated evoked glutamatergic transmission to IPN. The facilitatory effects of MOR activation on glutamate co-release was also observed from cholinergic-defined HP synapses. The potentiation of excitatory transmission mediated by MOR activation persisted in the presence of blockers of GABA receptors or voltage-gated sodium channels, suggesting a monosynaptic mechanism. Finally, disruption of MOR in the MHb abolished the faciliatory action of DAMGO, indicating that this non-canonical effect of MOR activation on excitatory neurotransmission at the HP synapse is dependent on pre-synaptic MOR expression. Our study demonstrates canonical inhibitory effects of MOR activation in somatodendritic compartments, but non-canonical faciliatory effects on evoked glutamate transmission at the HP synapse, establishing a new mode by which MOR can modulate neuronal function.

neuroscience↗

bioRxiv2024

Deep homology of a brachyury regulatory syntax and origin of the notochord

The brachyury gene encodes a T-box transcription factor (TF) that is crucial for the development of the notochord, a novel trait of chordates1. Brachyury expression in axial mesodermal cells (notochord progenitors) is regarded as a chordate innovation2, yet it remains unclear how the chordate ancestor acquired this expression domain. By examining the sequences of previously identified notochord enhancers of several chordate brachyury genes3-5, we uncovered a regulatory syntax consisting of binding sites for four TFs (Su(H), Foxh1, Zic, and Ets) with a strict order and orientation. We also identified this syntax, here named SFZE, in potential cis-regulatory modules (CRMs) of brachyury orthologs in various non-chordate animals and even in Capsaspora, a close unicellular relative to animals. Reporter assays demonstrated that SFZE-containing CRMs from non-chordate organisms exhibited regulatory activity in the zebrafish notochord, and mutagenesis of the TF sites reduced the activity. Furthermore, the SFZE syntax in sea urchin confers its endoderm activity, with the TF sites functionally decoupled during gastrulation. These findings indicate that the association of the SFZE syntax with brachyury is ancient, likely predating the origin of animals. The emergence of axial brachyury expression is therefore probably not the result of a newly acquired notochord enhancer, but is instead likely attributed to co-option of upstream signals acting on the conserved SFZE syntax, which facilitates the origin of the notochord from rudimentary endodermal cells.

evolutionary biology↗

bioRxiv2024

CENcyclopedia: Dynamic Landscape of Kinetochore Architecture Throughout the Cell Cycle

The kinetochore, an intricate macromolecular protein complex located on chromosomes, plays a pivotal role in orchestrating chromosome segregation. It functions as a versatile platform for microtubule assembly, diligently monitors microtubule binding fidelity, and acts as a force coupler. Comprising over 100 distinct proteins, many of which exist in multiple copies, the kinetochores composition dynamically changes throughout the cell cycle, responding to specific timing and conditions. This dynamicity is important for establishing functional kinetochores, yet the regulatory mechanisms of these dynamics have largely remained elusive. In this study, we employed advanced quantitative immunofluorescence techniques to meticulously chart the dynamics of kinetochore protein levels across the cell cycle. These findings offer a comprehensive view of the dynamic landscape of kinetochore architecture, shedding light on the detailed mechanisms of microtubule interaction and the nuanced characteristics of kinetochore proteins. This study significantly advances our understanding of the molecular coordination underlying chromosome segregation.

cell biology↗

bioRxiv2024

Assessing RNA velocity stability across syntheticreplicates using count splitting

MotivationRNA velocity is a computational framework that predicts future cell states from single-cell RNA sequencing data, offering valuable insights into dynamic biological processes. However, there is a lack of general methods to quantify the uncertainty and stability of these predictions from various RNA velocity methods. ResultsWe present a novel framework for evaluating RNA velocity stability using negative binomial count splitting to generate independent data replicates, a metric we call replicate coherence. Testing five RNA velocity methods across datasets for mouse erythroid, pancreatic, and human brain development, we identify significant performance differences and inconsistencies, such as reversed velocity flows. Our framework remains robust even when intermediary cell states are missing. Furthermore, we introduce a signal-to-random coherence metric to guide model selection. We demonstrate that selecting fits with high replicate coherence uncovers more biologically informative gene pathways. This broadly applicable approach provides a rigorous tool for assessing and comparing RNA velocity methods across diverse biological contexts. Availability and implementationThe code and analyses are available at https://github.com/linnykos/veloUncertainty.

genomics↗