Biology subjects

Chen, G.

Publications and source records attributed to Chen, G..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Bi-modal microwave neuromodulation via thermal and nonthermal mechanisms

Electrical neuromodulation, the current clinical standard, is invasive, expensive, and prone to malfunction. Electromagnetic waves can perform noninvasive neuromodulation, but existing methods are limited by the tradeoff between penetration depth and spatial precision. Microwaves in the 0.9 - 3 GHz range are widely used for telecommunications and can penetrate to the deep brain. Microwaves have been shown to nonthermally modulate neural activity, but the acute bioeffects remain unclear and under-studied. Here, we employ a microwave rod antenna (MRA) to demonstrate bi-modal neuromodulation via thermal and non-thermal mechanisms. The MRA enabled electrophysiological recordings of neurons exposed to microwaves, which elucidated the differential effects of pulsed and continuous microwaves on neurons. These findings build the foundation for developing microwave-based wireless neuromodulation devices for drug-free treatment of seizures and chronic pain.

neuroscience↗

bioRxiv2025

QuickProt: A Fast and Accurate Homology-Based Protein Annotation Tool for Non-Model Organism Genomes

Despite the increasing number of genomes, the annotation of protein-coding genes is still lacking. In some genomes where transcriptome data is not available, various tools have been developed to annotate genes based on homologous proteins, but each tool has its limitations. Here, we propose quickprot, a user-friendly command-line tool that constructs a set of redundancy-free gene models from the target genome based on the homologous protein sequences of a set of closely related species. We tested its applicability in stonefish, and the results showed that it has high accuracy and runs faster than existing tools. This low-cost, high-precision annotation method is expected to become a useful tool for genome annotation, which will contribute to the research of comparative genomics. The software is implemented in Python and licensed under the MIT License. The source code, documentation, and tutorials can be obtained at https://github.com/thecgs/quickprot.

bioinformatics↗

bioRxiv2025

Enzyme Co-Scientist: Harnessing Large Language Models for Enzyme Kinetic Data Extraction from Literature

The extraction of molecular annotations from scientific literature is critical for advancing data-driven research. However, traditional methods, which primarily rely on human curation, are labor-intensive and error-prone. Here, we present an LLM-based agentic workflow that enables automatic and efficient data extraction from literature with high accuracy. As a demonstration, our workflow successfully delivers a dataset containing over 91,000 enzyme kinetics entries from around 3,500 papers. It achieves an average F1 score above 0.9 on expert-annotated subsets of protein enzymes and can be extended to the ribozyme domain in fewer than 3 days at less than $90. This method opens up new avenues for accelerating the pace of scientific research.

biochemistry↗

bioRxiv2025

Benchmarking Pre-trained Genomic Language Models for RNA Sequence-Related Predictive Applications

RNA plays a pivotal role in diverse cellular functions across organisms. Developing computational algorithms for RNA sequence related questions is highly valuable. Recently, genomic language models (gLMs) with pre-training have emerged, offering flexibility for various downstream prediction tasks. However, comprehensive and fair evaluations of gLMs are lacking. In this study, we benchmark eight gLMs on prediction tasks covering four RNA processes, highlighting their strengths and limitations. While gLMs excel in performance overall, the larger model is not always better. Interestingly, models that integrate biological information consistently perform well in related tasks. Notably, gLMs demonstrate superior performance with limited training data, whereas task-specific methods achieve comparable performance with better computational efficiency when sufficient training data is available. Finally, we provide recommendations for model selection in different scenarios. These evaluation results underscore the potential of gLMs and suggest areas for future improvement.

genomics↗

bioRxiv2025

CRISPR-based shuttle cloning of 1,397 human genes into UAS vectors

The construction of genome-wide DNA libraries from publicly available resources is essential for leveraging functional genomics to investigate complex biological systems. However, all existing high-throughput cloning methods for transferring DNA fragments between vectors require PCR amplification of the DNA fragments, rendering the construction of genome-wide DNA libraries labor-intensive and time-consuming. By introducing a concept of CRISPRshuttle cassette, we herein present a method named CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method enables the high-throughput transfer of numerous DNA fragments from original plasmids with identical backbones to a different vector background without the need for PCR amplification of the DNA fragments. The procedure comprises two-step test tube reactions followed by bacterial transformation. Using CRISPRshuttle we successfully generated a library of GAL4/UAS-based UAS-ORF plasmids covering 1,397 human genes conserved in Drosophila. This library may serve as a valuable resource for gain-of-function screening in cultured cells and for the creation of a transgenic UAS-ORF library in Drosophila.

molecular biology↗

bioRxiv2025

Intermittent Fasting Reprograms Chromatin Accessibility to Modulate Gene Expression in Brain and Muscle

Intermittent fasting (IF), a dietary regimen that mimics the natural feeding patterns observed across diverse organisms--from single-celled life to mammals--is thought to activate systemic survival responses and confer health benefits. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, we utilized ATAC-Seq and RNA-Seq to investigate how IF influences chromatin accessibility and gene expression in brain and muscle tissues of mice, compared to ad libitum feeding. Our results reveal that IF induces significant changes in chromatin accessibility, modulating pathways related to metabolism, ribosome function, HIF-1 signaling, and glycolysis. Motif analysis identified tissue-specific transcription factors enriched in IF-regulated regions, including Sp1, Mef2a, NeuroD2, Banp, and NFIA in the brain, and SMAD4, TCF4, STAT5B, NKX3-1, and ZEB2 in muscle. Integrative analysis of ATAC-Seq and RNA-Seq data demonstrated that IF upregulates 50 genes and downregulates 15 in the cortex, while upregulating 31 genes and downregulating 10 in muscle. These gene expression changes are linked to pathways associated with neuroprotection and enhanced muscle function, offering mechanistic insights into the health benefits of IF. Our findings underscore the role of IF-induced chromatin remodeling in driving adaptive gene regulation.

bioinformatics↗

bioRxiv2025

scKAN: Interpretable Single-cell Analysis for Cell-type-specific Gene Discovery and Drug Repurposing via Kolmogorov-Arnold Networks

Single-cell analysis has revolutionized our understanding of cellular heterogeneity, yet current approaches face challenges in efficiency and interpretability. In this study, we present scKAN, a framework that leverages Kolmogorov-Arnold Networks for interpretable single-cell analysis through three key innovations: efficient knowledge transfer from large language models through a lightweight distillation strategy; systematic identification of cell-type-specific functional gene sets through KANs learned activation curves; and precise marker gene discovery enabled by KANs importance scores with potential for drug repurposing applications. The model achieves superior performance on cell-type annotation with a 6.63% improvement in macro F1 score compared to state-of-the-art methods. Furthermore, scKANs learned activation curves and importance scores provide interpretable insights into cell-type-specific gene patterns, facilitating both gene set identification and marker gene discovery. We demonstrate the practical utility of scKAN through a case study on pancreatic ductal adenocarcinoma, where it successfully identified novel therapeutic targets and potential drug candidates, including Doconexent as a promising repurposing candidate. Molecular dynamics simulations further validated the stability of the predicted drug-target complexes. Our approach offers a comprehensive framework for bridging single-cell analysis with drug discovery, accelerating the translation of single-cell insights into therapeutic applications.

bioinformatics↗

bioRxiv2025

p.D372H: A novel SCN5A mutation associated with Brugada syndrome

BackgroundBrugada syndrome (BrS) is a genetic cardiac arrhythmia disorder inherited in an autosomal dominant manner, characterized by ST-segment elevation in the right precordial leads (V1-V3) on electrocardiograms (ECGs). This syndrome predominantly affects young individuals with structurally normal hearts and significantly increases the risk of ventricular arrhythmias and sudden cardiac death (SCD). The most common genotype found among BrS patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+) channel (Nav1.5) by different mechanisms. MethodsPlasmids containing SCN5A were constructed using PCR and site-directed mutagenesis to create the D372H mutation. HEK293 cells were cultured and transfected with the wild-type and mutant constructs. Patch-clamp recordings assessed sodium current characteristics. Confocal microscopy visualized channel localization. Quantitative RT-PCR analyzed mRNA expression levels, while Western blot evaluated protein expression using specific antibodies. We identified a novel missense mutation, D372H, in the SCN5A gene associated with Brugada syndrome. Functional assays in HEK293 cells expressing the D372H mutant revealed a near-complete loss of sodium currents. Subsequent experiments with co-transfection of WT and D372H plasmids demonstrated that co-expression led to a significant reduction in current density in WT-expressing cells (P < 0.05). The D372H mutation also resulted in a hyperpolarizing shift of approximately 20 mV in the voltage dependence of inactivation, while activation and recovery kinetics remained unaffected. Additionally, confocal microscopy showed reduced membrane localization of the D372H mutant, with a significant decrease in protein expression levels confirmed by Western blot and RT-qPCR analyses. ConclusionIn summary, our findings indicate that the D372H mutation in the Nav1.5 sodium channel leads to significant reductions in sodium current density, altered channel expression, and impaired membrane localization. These changes contribute to the pathophysiology of Brugada syndrome by disrupting cardiac action potential dynamics.

genetics↗

bioRxiv2025

Integrating transcriptomic data with metabolic model unravels the electron transfer mechanisms of Methanosarcina barkeri

Methanogenic archaea, particularly Methanosarcina, are pivotal to the global carbon cycle and renewable energy production due to their versatile metabolic capabilities. Although transcriptomic analysis is widely employed to identify key genes and pathways in Methanosarcina under various methanogenic conditions--including the emerging direct interspecies electron transfer (DIET)-based methanogenesis--the weak correlation between gene expression levels and protein abundances poses challenges for interpreting transcriptomic data. To address this, we integrated transcriptomic data into a metabolic model of Methanosarcina barkeri for the first time, enabling more refined predictions and enhancing the interpretability of transcriptomic insights. This novel integrated model was subsequently utilized to simulate aceticlastic, hydrogenotrophic, and DIET-based methanogenesis. The results revealed that previous assumptions failed to account for the role of the CO2 reduction pathway in aceticlastic methanogenesis. The model also successfully captured key transcriptomic features of DIET-based methanogenesis, clarifying the functional roles of crucial enzymes like the F420H2 dehydrogenase Fpo and the transmembrane hydrogenase Vht in electron transfer. This integrative approach provided a deeper understanding of electron transfer mechanisms in M. barkeri and offered valuable insights for advancing methanogen-based biotechnologies. Moreover, the study critically evaluated the relationship between gene expression and metabolic flux, establishing a practical framework for deriving meaningful insights from the growing volume of transcriptomic data.

bioengineering↗

bioRxiv2025

Complete genome sequence assembly elucidates evolution and regulation of 5S rDNA loci in the greater duckweed Spirodela polyrhiza

We resolved the molecular architecture of the 5S rDNA loci in an aquatic monocot Spirodela polyrhiza. Measurements of fluorescence in situ hybridization signals revealed two loci with 5S rDNA clusters. A combination of the extra-long DNA reads and conventional sequencing of 5S rDNA repeats allowed the assembling of complete loci sequences located on ChrSp6 and ChrSp13. The homologous chromosomes of the ChrSp6 locus contain clusters of 40 and >60 copies of 5S rDNA repeat units with intergenic spacer (IGS) of 400 bp. The ChrSp13 locus is represented by 5S rDNA clusters of rRNA genes spaced by IGSs of 1,056 or 1,069 bp arranged in two sub-clusters, suggesting a different rate of repeat homogenization between loci. The G/C-rich 5S rDNA arrays in both loci are embedded in A/T-enriched chromosome regions with possible regulatory functions. The TATA-like boxes of the 5S rDNA repeat on ChrSp6 and ChrSp13 exhibit different affinities for the TATA-binding protein in 3D modeling of protein/DNA interactions, suggesting a locus-specific regulation of rRNA transcription. Our findings shed light on the molecular architecture of the 5S rDNA loci, which could foster an innovative era for the principles of evolution and regulation of rRNA genes in plants.

molecular biology↗

bioRxiv2025

Multicilia dynamically transduce Shh signaling to regulate choroid plexus functions

Choroid plexus is a major site for cerebrospinal fluid (CSF) production, characterized by a multiciliated epithelial monolayer that regulates CSF production. We demonstrate that defective choroid plexus ciliogenesis or Intraflagellar transport yields neonatal hydrocephalus, at least in part, due to increased water channel Aqp1 and ion transporter Atp1a2 expression. We demonstrate choroid plexus multicilia as sensory cilia, transducing both canonical and non-canonical Shh signaling. Interestingly, it is the non-canonical Shh signaling that represses Aqp1 and Atp1a2 expression by Smo/Gi/cAMP pathway. Choroid plexus multicilia exhibit unique ciliary ultrastructure, carrying features of both primary and motile cilia. Unlike most cilia that elongate during maturation, choroid plexus ciliary length decreases during development, causing a decline of Shh signaling intensity in developing choroid plexus, a derepression of Aqp1 and Atp1a2, and ultimately, an increased CSF production. Hence, developmental dynamics of choroid plexus multicilia dampens the Shh signaling intensity to promote CSF production.

developmental biology↗

bioRxiv2025

Genome diversity and phylogeny of the section Alatae of genus Lemna (Lemnaceae), comprising the presumed species Lemna aequinoctialis, Le. perpusilla and Le. aoukikusa

O_LIThe Section Alatae of genus Lemna of the monocotyledonous aquatic duckweed family (Lemnaceae) consists of rather diverse accessions with unknown phylogeny and unclear taxonomic assignment. In contrast to other duckweeds, some Alatae accessions, in addition to mainly vegetative propagation, produce readily flowers and viable seeds. We analyzed the genomic diversity and phylogenetic relationship of 52 Alatae accessions. C_LIO_LIFor this purpose, we applied multiple molecular and cytogenetic approaches, including plastid and nuclear sequence polymorphisms, chromosome counting, genome size determination and genomic in situ hybridization in combination with geographic distribution. C_LIO_LIWe uncovered ploidy variation, recurrent hybridization, and backcrosses between diploid species and their hybrids. The latter successfully spread over three continents. C_LIO_LIThe results elucidate the evolution of Alatae accessions (summarized in Fig. 7) and explain the difficult taxonomic assignment of distinct accessions. Our study might be an example for analogous studies to resolve the hitherto unclear relationships among accessions of the duckweed genera Wolffiella and Wolffia. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/632899v1_fig7.gif" ALT="Figure 7"> View larger version (23K): org.highwire.dtl.DTLVardef@d05f08org.highwire.dtl.DTLVardef@f33c5org.highwire.dtl.DTLVardef@ff7adorg.highwire.dtl.DTLVardef@4da6e4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7C_FLOATNO Hypothetical scenario of evolution within Lemna Section Alatae. In bold are accessions from America. The classically defined diploid species Le. aequinoctialis and Le. perpusilla sensu stricto were confirmed by the applied molecular methods. The previously named Le. aoukikusa accessions were uncovered as tetraploid interspecific hybrids between Le. aequinoctialis and Le. perpusilla. In addition, further tri- and tetraploid hybrids as well as tri- and tetraploid "Backcross hybrids" were detected from the USA and China, respectively. C_FIG

evolutionary biology↗

bioRxiv2025

TOC1 phosphorylation disproportionally enhances chromatin binding at rhythmic gene promoters

Protein phosphorylation is a key regulatory mechanism in circadian systems. TIMING OF CAB EXPRESSION 1(TOC1) is a core transcriptional repressor in the plant circadian system that is phosphorylated near its N-terminus. Phenotype testing of single and multi-phosphosite mutants shows incomplete rescue of the short period toc1 mutant. We establish that TOC1 phosphorylation (particularly at S175) is necessary for optimal interaction with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and PHYTOCHROME INTERACTING FACTOR 5 (PIF5) at the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter to downregulate CCA1 expression. At the same time, expression of the closely related LATE ELONGATED HYPOCOTYL (LHY) also requires TOC1, but is independent of the TOC1 phosphorylation state, suggesting different TOC1-dependent mechanisms in the repression of these two genes. We additionally show how phosphorylation-dependent interactions of TOC1 at specific clock gene promoters selectively regulate these circadian system components more acutely than non-rhythmic genes. Our genome-wide analysis shows that the phosphostate of TOC1 is important for optimal chromatin presence and robust rhythmic gene expression.

plant biology↗

bioRxiv2025

Vat Photopolymerization of Porous Scaffolds: Stabilization and Layer Thickness Control for Micron-Scale Accuracy

Vat Photopolymerization (VPP) holds much promise for producing biomaterial constructs such as porous scaffolds. However, achieving micron-scale pore dimensions with precision presents a challenge. This study offers an innovative approach to stabilize the silicone elastomer vat surface permitting micron-scale layer thickness accuracy to be maintained. Internal and surface contamination on the poly(dimethyl siloxane) (PDMS) vat surface were observed and effectively controlled with a pre-saturation methodology, and porous structures with cubical pores were then printed with varying layer thicknesses. These structures demonstrate the ability to achieve micrometric resolution and layer thicknesses as fine as 32 {micro}m. A scaffold suitable for in vivo implantation with 40 {micro}m cubical pores was successfully printed within 5 hours using a stabilized PDMS vat surface. Additionally, the methodologys adaptability to intricate non-linear edge porous structures underscores its versatility across X, Y, and Z-axis.

bioengineering↗

bioRxiv2024

The Basal Ciliary but not Cytosol PKA Specifically Regulates HH Pathway Downstream of Smoothened

Effectors of the vertebrate Hedgehog (HH) signaling pathway are organized through primary cilia (PC) that grow and retract in lockstep with the cell cycle in response to extracellular signals. Protein kinase A (PKA), a kinase with ubiquitous distribution in most cells, functions as a specific negative regulator of the HH pathway. Its functional specificity in the HH pathway has been suggested to be controlled by cAMP in the PC. However, the regulation of PKA and its functions in PC remain unclear, in part due to the lack of observation of PKA localization in PC during HH resting state as well as conflicting reports of the dynamic changes of cAMP in cilia and HH pathway activity. To address this issue, we have developed a ciliary-localized FRET-based A-kinase activity probe (Nphp3N-AKAR2-CR) as an improved biosensor for monitoring real-time PKA activity in the PC of both cultured cells and living zebrafish embryos. Although the PKA catalytic subunit (PKA-C) was not observed in PC, basal PKA activity in cells could be detected with this probe. In addition, we have found that only ciliary-targeted PKA and not cytosolic PKA, can modulate the HH pathway, even when the integrity of the PC is disrupted. Notably, ciliary PKA activity was barely changed either by inhibition or activation of the HH pathway at the level of Smoothened (SMO), the obligate HH signal transducer. Moreover, we found that even low concentration of the adenylyl cyclase agonist forskolin (FSK) can efficiently inhibit the HH pathway in the presence of the constitutively active variant SMOA1, suggesting that the activation of the HH pathway by SMO may not be due solely to direct regulation of PKA activity in the PC.

cell biology↗

bioRxiv2024

Circumferential actomyosin bundles drive endothelial cell deformations to constrict blood vessels

Following the formation of new blood vessels, vascular remodelling ensues to generate a hierarchical network of vascular tubes with optimal connections and diameters for efficient blood perfusion of tissues. How transitions in endothelial cell (EC) number and shape are coordinated to define vessel diameter during development remains an open question. In this study, we discovered EC deformations, rearrangements and transient formation of self-seam junctions as key mechanisms that explain a negative relationship between cell number and vessel diameter. High-resolution analysis of actin cytoskeleton organization disclosed the generation of tension-bearing, circumferential actomyosin bundles in the endothelial cortex that drive EC deformation and vessel constriction. Importantly, the loss of circumferential actin bundles in krit1/ccm1-deficient ECs causes cell enlargement and impaired vessel constriction that culminate in dilated vessels, characteristic of cerebral cavernous malformation. Our multiscale study therefore underpins circumferential actomyosin-driven EC deformations in controlling vessel size and in the prevention of vascular malformations.

cell biology↗

bioRxiv2024

AMPK acts to remove immune barriers to CD8+ T cell-mediated immunity against hepatocellular carcinoma

Dysregulated metabolism in tumor tissues, and para-tumor tissues alike, can lead to immunosuppression, which may underlie cancer development. However, metabolic intervention as a therapeutic strategy has been of no avail. In this study, we explored the anti-cancer therapeutic effect of aldometanib that specifically targets lysosome-associated aldolase to mimic glucose starvation and thereby activates the lysosomal AMP-activated protein kinase (AMPK), a master regulator of metabolic homeostasis. We show that aldometanib inhibits the growth of HCC in an AMPK-dependent manner, allowing hepatoma-bearing mice to survive to mature ages, although aldometanib does not possess cytotoxicity towards HCC or normal cells. Intriguingly, aldometanib exerts anti-cancer effects only in immune-competent host mice, but not in immune-defective mice. We have further found that the HCC tissues in aldometanib-treated mice are massively infiltrated with CD8+ T cells, which is not seen in mice with liver-specific knockout of AMPK. Our findings thus suggest that the metabolic regulator AMPK rebalances the tumor microenvironment to allow the cytotoxic immune cells inside the body to eliminate cancer cells and effectively contain the tumor tissues. The finding that metabolic intervention can make cancer a life-long manageable disease, may potentially usher in a new era of cancer therapy.

immunology↗

bioRxiv2024

Oligomerization of PRMT1 Regulated RNA-Binding Protein Cascade Promotes Pancreatic Ductal Adenocarcinoma

PRMT1 is the predominant type I protein arginine methyltransferase responsible for generating monomethylarginine and asymmetric dimethylarginine (MMA and ADMA) in various protein substrates. It regulates numerous biological processes, including RNA metabolism, mRNA splicing, DNA damage repair, and chromatin dynamics. The crystal structure of rat PRMT1 has been previously reported as a homodimer; however, higher-order oligomeric species of human PRMT1 have been observed in vivo, and their structural basis remains elusive. In this study, we present the cryo-EM structure of human PRMT1 in its oligomeric form, revealing novel interfaces crucial for PRMT1 self-assembly and normal function. PRMT1 shows a strong preference for intrinsically disordered RGG/RG motifs, which are commonly found in RNA-binding proteins (RBPs), highlighting its critical role in regulating RNA metabolism. In vitro studies indicate that disrupting PRMT1 oligomerization impairs its binding affinity for RGG motif substrates, thereby reducing arginine methylation levels on these substrates. This finding emphasizes the essential role of the oligomeric state of PRMT1 in its function with RGG motif-containing RBPs. In vivo, the loss of PRMT1 oligomerization leads to decreased global ADMA levels in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits PDAC tumor growth. Collectively, we propose that PRMT1 oligomerization, rather than mere dimerization, is sufficient for PRMT1-driven PDAC tumor growth, offering novel insights into the potential therapeutic targeting of PRMT1 oligomeric forms in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/627716v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1ed6232org.highwire.dtl.DTLVardef@1c9e688org.highwire.dtl.DTLVardef@3e2c43org.highwire.dtl.DTLVardef@1cbf33e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗