Biology subjects

Kim, D.

Publications and source records attributed to Kim, D..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Computational design of cysteine proteases

Despite advances in de novo enzyme design, success has been largely limited to low energy barrier model reactions. Amide bonds such as those linking amino acids along the peptide backbone are stable for hundreds of years in neutral aqueous solution because of the high energy barrier to hydrolysis. Here we describe the de novo design of enzymes which utilize an activated cysteine nucleophile to hydrolyze the polypeptide backbone in a sequence-dependent manner, with a success rate of 13/69=19% and rate enhancements over the background reaction (kcat/kuncat) of up to 3 x 10^7. The designed proteases have folds very different from proteases in nature (TM score < 0.50), and six crystal structures are very close to the design models (Ca RMSDs < 1.2 A), highlighting the capacity for generalization and the accuracy of the design methodology. Experimental and computational analyses suggest that the remaining gap in activity to the most active native cysteine proteases arises from imperfections in active site preorganization and substrate positioning. The designed proteases efficiently cleave their targets in mammalian cells, opening the door to a wide range of synthetic biology applications.

biochemistry↗

bioRxiv2025

Computational design of metalloproteases

Although significant progress has been made in creating de novo metalloenzymes that hydrolyze activated esters, the energetically demanding cleavage of amide bonds has remained a major challenge for enzyme design: amide bonds are significantly more stable than ester bonds, the amine leaving groups in proteins are not activated, and peptide substrates are flexible making them difficult to bind precisely. Here, we report the de novo design of zinc proteases from minimal catalytic motifs using RoseTTAFold Diffusion 2 for Molecular Interfaces, optimized for both enzyme and protein-protein interaction design. Of 135 computational designs experimentally tested, 36% had activity and cleaved precisely at the intended site. The most active design accelerates peptide bond hydrolysis by more than 10^8-fold relative to the uncatalyzed reaction, and by over 10^10-fold following the introduction of four point mutations that enhanced active-site preorganization4. Building on these capabilities, we designed metalloproteases that specifically cleave human TDP-43, the amyloid-{beta} peptide, and serum amyloid A with rate accelerations up to 9.2x10^8-fold over background. We illustrate the potential of our approach for bio-orthogonal control over cell state by generating caged cytokines and caged receptor antagonists that are selectively unmasked by our designed proteases. These results demonstrate that de novo enzyme design has advanced well beyond model reactions with activated substrates and open the door to design of proficient metallohydrolases for a wide range of applications in medicine and bioremediation.

biochemistry↗

bioRxiv2025

PAF15 stabilizes PCNA on DNA and slows down its sliding dynamics

The PCNA-associated factor 15 (PAF15) protein is essential for human genome stability by regulating DNA replication and repair through its interaction with proliferating cell nuclear antigen (PCNA). Despite its significance, the mechanistic details of the PAF15-PCNA interaction remain insufficiently understood. Here, we reveal how PAF15 influences the sliding dynamics and function of PCNA. Using single-molecule diffusion analysis on a DNA skybridge platform, we show that PAF15 binding stabilizes PCNA in its DNA-bound state, reduces its diffusion rate along the duplex, restricts accessibility to PCNA PIP-box binding sites, and regulates PCNA loading and unloading on DNA via replication factor C (RFC). Our results are consistent with its coordinating role in both high-fidelity replication and lesion bypass. These findings establish PAF15 as a key regulator of PCNA function, serving as a mobile platform for DNA-editing enzymes and thereby influencing genome maintenance.

biophysics↗

bioRxiv2025

Large Language Models for Accessible Reporting of Bioinformatics Analyses in Interdisciplinary Contexts

Health and life scientists frequently rely on quantitative experts to perform complex data analyses, yet interpretation of these results often becomes a bottleneck due to communication barriers across disciplines. Large Language Models (LLMs) have the potential to act as intermediaries that translate analytical outputs into accessible scientific narratives, but their ability to reliably interpret outputs from real-world biomedical data analyses across disciplines remains unclear. In this study, we investigate how state-of-the-art LLMs behave when integrated into real bioinformatics analysis workflows as report-generation assistants for interdisciplinary teams. We evaluated LLM-generated summaries using both automated and human evaluation frameworks to ensure holistic evaluation. Automated assessment employed multiple choice questions designed using Bloom's taxonomy to assess multiple levels of understanding, while human evaluation tasked scientists to score summaries for factual consistency, lack of harmfulness, comprehensiveness, and coherence. All generally produced readable and largely safe summaries, confirming their value for first-pass translation of technical analyses, however frequently misinterpreted visualisations, produced verbose summaries and rarely offered novel insights beyond what was already contained in the analytics. Our findings suggest that LLMs are best suited for easing interdisciplinary communication rather than replacing domain expertise and human oversight remains essential to guarantee accuracy, interpretative depth, and the generation of genuinely novel scientific insights.

bioinformatics↗

bioRxiv2025

When Animals Turn Inside Out: The Eversion of Bloodworms

Bloodworms, Glycera dibranchiata, possess an eversible proboscis that normally remains concealed within their bodies but explosively everts if the worm attacks or burrows. How does the bloodworm evert quickly and reliably? In a series of experiments, we characterize bloodworm kinematics, pressure, and material properties to estimate the criteria for eversion safely without rupture of the proboscis. We predict the proboscis can withstand pressures 50 times higher and bending strains up to three times higher than the respective values observed. We also present a dimensional analysis of eversion, finding that everting animals, from frogs to snails to sharks, do not satisfy Froudes law for equivalence of velocities. Our findings may help inspire the development of pressure-driven soft robots with efficient retraction capabilities.

biophysics↗

bioRxiv2025

CRISPR-Induced NUT Suppression Promotes Differentiation and Enhances Trop2-Targeted Immunocytokine Response in NUT Carcinoma

NUT carcinoma (NC) is an aggressive malignancy driven by NUTM1 gene rearrangements with limited therapeutic options. Here, we show that direct suppression of NUTM1 using CRISPR/Cas9 induces squamous-like differentiation and upregulates TROP2 expression in NC cells. Building on this finding, we developed a TROP2-interferon beta (IFN-{beta}) mutein immunocytokine that selectively targets TROP2-expressing tumors. Combined NUTM1 suppression and TROP2-targeted immunotherapy synergistically enhanced cytotoxic and immune-mediated responses in vitro. Transcriptomic and spatial analyses of NC patient tumors revealed that differentiation status correlates with TROP2 expression, upregulated immune pathways, and favorable clinical outcomes. Our results suggest that overcoming differentiation blockade not only alters tumor phenotype but also creates a more immune-permissive microenvironment. These findings highlight the therapeutic potential of sequential tumor reprogramming followed by targeted immunotherapy in treating NC and propose a broader strategy for overcoming differentiation blockade in fusion-driven cancers.

cancer biology↗

bioRxiv2025

Engineering a Biomimetic Multiphasic Suture Anchor System for Enhanced Rotator Cuff Enthesis Regeneration

Conventional suture anchor methods in rotator cuff repair often fail to replicate the native tendon-to-bone interface, leading to re-tears due to stress concentrations and poor biological integration at anchor sites. To address these challenges, we engineered a biomimetic multiphasic scaffold system (BMS) that integrates with standard suture anchors and deliver spatially organized structural and biological cues to enhance enthesis regeneration. The BMS comprises three distinct phases: aligned nanofibrous decellularized bovine Achilles tendon extracellular matrix (dECM) with stiff methacrylated hyaluronic acid (MeHA) for tendon regeneration; nonaligned nanofibrous dECM with soft MeHA for fibrocartilage formation; and a porous, citrate-based composite scaffold with bioactive glass for bone integration. In vitro, the BMS facilitated zone-specific tenogenic, fibrochondrogenic, and chondrogenic differentiation. Further, in vivo, it promoted successful integrative healing, forming distinct tendon, fibrocartilage, and bone regions at the repair site. This advanced multiphasic scaffold replicates native tissue properties, offering a promising strategy to improve rotator cuff repair. Its integration with conventional suture anchors provides an innovative design that enhances mechanical fixation and guides enthesis healing to reduce re-tear rates. Broadly, this platform offers a versatile solution for biointegrative repair strategies across complex soft-to-hard tissue interfaces.

bioengineering↗

bioRxiv2025

Organelle-Aware Representation Learning Enables Label-Free Detection of Mitochondrial Dysfunction in Live Human Neurons

Mitochondrial dysfunction is a convergent hallmark of neurodegenerative diseases and represents a promising biomarker for early diagnosis and therapy. However, current in vitro assays rely on fluorescence or electron microscopy, which are invasive, low-throughput, and incompatible with longitudinal analysis. Here, we present a noninvasive framework by integrating label-free optical diffraction tomography (ODT) with organelle-aware representation learning to detect subtle mitochondrial dysfunction in live human induced pluripotent stem cell (hiPSC)-derived neurons. Through virtual staining of the nuclei, lysosomes, and mitochondria, we establish two complementary and interpretable classification pipelines: a deep learning model with organelle-aware encoder and a logistic regression model on morphometric descriptors. Both models achieve approximately 85% accuracy: the deep model provides end-to-end prediction with minimal feature engineering, whereas the logistic regression model offers a more interpretable, feature-based approach. To our knowledge, this is the first demonstration of ODT-based organelle-resolved virtual staining in live human neurons, establishing a scalable, non-invasive platform for mitochondrial disease modeling, drug discovery, and neurodegeneration research.

neuroscience↗

bioRxiv2025

AGO1 in neural progenitor cells orchestrates brain development and sociability via LIN28A-REELIN axis

AGO1, an essential RNA binding protein (RBP) in RNA interference, is associated with autism spectrum disorder (ASD). However, the precise functions of AGO1 in brain development and related disorders remain largely unexplored. Here, we report the critical roles of AGO1 in neural progenitor cells (NPCs) in promoting sociability and shaping the structure of the developing brain. Ago1 knockout (KO) in the mouse brain leads to hyposociability, a characteristic symptom of ASD. In human forebrain organoids, AGO1 KO disrupts the formation of ventricle-like structures and delays cortical layer development. We discovered that AGO1 KO results in a loss of polarity in NPCs, which subsequently reduces neuronal development. These AGO1 KO phenotypes originate from the failure to suppress LIN28A in NPCs. AGO1 is primarily localized in the nucleus within NPCs and binds to the LIN28A promoter region, thereby inhibiting LIN28A transcription. We found that increased LIN28A in AGO1 KO NPCs reduces REELIN expression, a key regulator of brain development, by binding directly to the REELIN mRNA. The aberrant polarity phenotype in AGO1 KO NPCs was successfully ameliorated by either LIN28A knockdown or recombinant REELIN treatment. Collectively, our findings elucidate the intricate molecular mechanisms orchestrated by nuclear AGO1 in NPCs and underscores its pivotal roles in brain development, providing significant insights into ASD.

neuroscience↗

bioRxiv2025

Replicating without stress: a dengue replicon model reveals distinct host rewiring to accommodate persistence

Self-replicating viral RNAs, or replicons, are widely used to study virus-host interactions and screen antiviral compounds, yet their ability to model persistent infection and host adaptation remains poorly understood. Here, we characterize a stable subgenomic dengue virus serotype 2 (DENV2) replicon (DENV2-rep) that supports long-term viral RNA replication in human hepatoma cells without triggering cytotoxicity or canonical stress responses. Compared to live DENV2 infection, which induces ER stress and global translation shutdown, DENV2-rep cells maintain active translation and polysome integrity, while mounting an antiviral transcriptional response and resisting superinfection. Transcriptomic and proteomic profiling reveals that DENV2-rep cells accommodate long-term replication by remodeling of metabolic, secretory, and cytoskeletal pathways. In contrast, acute infection uncouples transcription from protein synthesis, limiting host proteome remodeling. We also detected marked depletion of the NS5 RNA polymerase in the DENV2-rep context. Together, these findings highlight host pathways that can be rewired to support persistent RNA replication and uncover post-translational regulation of viral proteins as a potential vulnerability. Our work clarifies the strengths and limitations of replicons as models for infection and reveals how persistent vRNA replication is tolerated through selective remodeling of translation, organelle composition, and protein stability.

microbiology↗

bioRxiv2025

The genomic complexity of invasion: cryptic lineages, founder effects, and polygenic sex determination in armored catfish

Genetic biocontrol approaches offer promising tools for managing invasive species. However, their success depends on species identity, demographic history, and the genomic architecture of sex determination, which remain poorly resolved for many non-model taxa. We integrated high-quality genome assembly, whole-genome resequencing, and population genomics to evaluate biocontrol feasibility in invasive suckermouth armored catfish (Loricariidae) in Texas. Mitochondrial phylogenies and genome-wide SNPs revealed cryptic diversity, with distinct lineages from at least two genera (Hypostomus and Pterygoplichthys) exhibiting strong drainage-level genetic differentiation. Effective population size estimates were exceptionally low, consistent with founder effects and moderate inbreeding. Genome-wide association analyses detected no large-effect loci for sex in either genus, despite high statistical power in Hypostomus, suggesting that sex determination is polygenic and/or environmentally influenced. These results identify key biological constraints for sex-ratio-based biocontrol and underscore the importance of genomic assessments prior to management interventions. More broadly, our study highlights how integrating genomic resources, demographic inference, and evolutionary context can guide invasive species control strategies.

genetics↗

bioRxiv2025

Sex chromosome turnover and mitonuclear conflict drive reproductive isolation

Identifying the genetic basis of reproductive barriers is essential for understanding the origin and maintenance of biological diversity. While some hybrid incompatibilities evolve as incidental byproducts of divergence1-3, those involving sex chromosomes and mitochondrial-nuclear interactions may arise through predictable pathways shaped by genomic conflict4-8. Yet, the extent to which such interactions drive the evolution of reproductive barriers and speciation in natural populations remains unclear9-11. Here, we use whole-genome resequencing in North American fishes to show that two hybridizing species possess distinct, nonhomologous sex chromosomes. These chromosomes exhibit strong associations with sex, reduced introgression in natural hybrid zones, segregation distortion in backcrosses, and an enrichment of nuclear-encoded mitochondrial genes, indicative of sex-linked mitonuclear incompatibilities. We identify a third, distinct sex chromosome in another hybridizing species, indicating repeated sex chromosome turnover within the clade. Parental crosses and genomic analyses suggest that at least one of these transitions was driven by a recessive female-determining mutation, a rare empirical example of a theoretically predicted but seldom observed mechanism of sex chromosome evolution. Together, these results link genomic architecture to hybrid dysfunction and behavioral isolation, providing strong empirical support for long-standing predictions about the role of sex-linked and cytonuclear incompatibilities in speciation.

evolutionary biology↗

bioRxiv2025

Update on the reproduction and interpretation of DIANA fMRI

Three years ago, our group reported direct imaging of neuronal activity (DIANA) with high spatiotemporal resolution, but its reproducibility and signal origin remain controversial. Here, we report the results of our reproduction experiments of DIANA fMRI performed to date using forelimb electrical stimulation at various magnetic field strengths in anesthetized mice, along with the characteristics of DIANA signal, called the pseudo-steady state (PSS). Theoretical analysis and Bloch simulations demonstrated that the spatial location and temporal phase of PSS oscillations are primarily determined by frequency-offset, and that their spatiotemporal superposition can generate peak signals in specific regions at specific timing that closely resemble DIANA signals. These findings suggest that if PSS oscillations are the primary source of DIANA signals, it may be premature to interpret them as neuronal responses to sensory stimulation. Further studies are needed to clarify the relationship between DIANA signals and brain activation.

neuroscience↗

bioRxiv2025

Accelerating Insight Discovery in Large Biomedical Text with Scalable Processing Framework

Large language models are increasingly being used by dermatology professionals to support diagnostic investigation, patient education, and medical research. While these models can help manage information overload and improve efficiency, concerns persist regarding their accuracy and potential reliance on dubious sources. We introduce Quanta, a hybrid system that combines large language models with established evaluation metrics, such as cosine similarity, to enable efficient summarization and interpretation of curated research corpora. This methodology ensures that synthesized insights remain domain-specific and contextually relevant, thereby supporting clinicians and researchers in navigating the expanding digital landscape of dermatology literature. Deployed within an interactive chatbot, the tool delivers direct answers to user queries, provides cross-publication insights, and can suggest new directions for research. Comparative evaluations on benchmark datasets demonstrate improvements in accuracy, efficiency, and computational cost, with the curated document approach enhancing reliability and reducing misinformation risk.

bioinformatics↗

bioRxiv2025

Cross-fostering affects microglia and cell death in the hippocampus of female and male degu pups

Parental care is essential for social, behavioural, and neural development of offspring. Disruption of parental care, for example through parental separation, can negatively affect offspring development in rodents. While previous work has focused on maternal and paternal deprivation, the effects of cross-fostering, another form of parental-offspring instability, on brain development remain poorly understood. Such disruptions are thought to induce stress, which in turn can suppress neurogenesis and increases inflammation and apoptosis in the hippocampus, with effects that often differ between sexes. Given that degus (Octodon degus) are born precocial and form strong attachments with their parents early in life, this study aimed to investigate the effect of cross-fostering on hippocampal development in female and male degu pups. At postnatal day 8, degus were assigned to either control (pups remained with parents and littermates), partial cross-foster (one pup per litter was cross-fostered), or full cross-foster (the entire litter was cross-fostered) conditions. At weaning (5-weeks-old), offspring brains were collected for immunohistochemistry to examine dentate gyrus volume, density of pyknotic cells, density of immature neurons, and number and morphology of microglia. Both types of cross-fostering reduced hippocampal cell death in both sexes relative to controls, while no significant effects were observed in the dentate gyrus volume or density of immature neurons. Cross-fostering did not affect total microglia density in either sex however full cross-fostered females had fewer amoeboid microglia compared to female controls. Together, these findings indicate that cross-fostering affects hippocampal microglia and cell death in pups, potentially disrupting circuit refinement and plasticity during development, with effects that vary by cross-fostering type, sex, and hippocampal region.

neuroscience↗

bioRxiv2025

Population genomics of the endangered scaly-foot snail defines conservation units amid deep-sea mining threats in Indian Ocean vents

Hydrothermal vents in the Indian Ocean face imminent deep-sea mining, yet lack baseline high-resolution connectivity data for their endemic fauna. We present the first population genomic study of the endangered scaly-foot snail (Chrysomallon squamiferum), an iconic species distributed across three vent biogeographic provinces. Analysing 125 individuals from eight vent fields using 14 million single-nucleotide polymorphisms, we identify five genetic groups that warrant recognition as evolutionarily significant units. Demographic modelling reveals critical contributions from extinct or unsampled phantom populations to the contemporary genetic structure. Together with physical ocean modelling, we show that the deep currents drive asymmetric south-to-north gene flows, while transform faults act as dispersal barriers. We propose Longqi-Duanqiao fields on the Southwest Indian Ridge and Wocan field on the Carlsberg Ridge as two isolated populations prioritised for protection. Given its exceptional adaptations, wide distribution, and public recognition, the scaly-foot snail is well-positioned to serve as both a flagship and umbrella species to ensure the survival of a broader suite of vent biodiversity. As the International Seabed Authority finalises its Mining Code in 2025, our findings provide essential genomic evidence to mitigate the impacts of mining on genetic diversity and inform transboundary conservation strategies for this vulnerable ecosystem.

ecology↗

bioRxiv2025

MEG3 Enhances Survival of Developing Human Neurons with CLCN4-Linked Autophagy Impairment

Genetic variations in CLCN4, encoding the H+/Cl- exchanger CLC-4, are associated with human neurodevelopmental disorders with highly variable phenotypes. A lack of physiologically relevant models has hampered molecular understanding of pathogenic mechanisms. We now establish engineered brain organoid and neuronal cell systems to examine impacts of patient-relevant CLCN4 genetic variations. We find that CLCN4 variants reduced excitatory neuron numbers due to early-stage cell death, accompanied by altered endo-lysosomal dynamics and disrupted autophagic flux. Transcriptomic profiling showed significant downregulation of long non-coding RNA MEG3 in CLCN4-variant neurons. Restoring MEG3 expression is sufficient to rescue cellular defects and improve survival of CLCN4-variant neurons. These findings link CLCN4 dysfunction with impaired autophagy and neuronal cell death, highlighting MEG3 as a potential therapeutic target for neurodevelopmental disorders involving autophagic dysfunction.

neuroscience↗

bioRxiv2025

Membrane Tethering of Honeybee Antimicrobial Peptides in Drosophila Enhances Pathogen Defense at the Cost of Stress-Induced Host Vulnerability

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics in combating multidrug-resistant pathogens, yet their clinical translation is hindered by proteolytic instability, cytotoxicity, and poor bioavailability. Here, we demonstrate that glycosylphosphatidylinositol (GPI)-mediated membrane tethering of honeybee defensin1 (Def1) in Drosophila melanogaster enhances its antimicrobial efficacy by [~]100-fold compared to secreted or untethered forms, while preserving physiological and behavioral integrity under baseline conditions. Using a genetically engineered Drosophila model, we expressed three Def1 variants: native (Def1), secreted (s-Def1), and membrane-tethered (t-Def1). Flies expressing t-Def1 exhibited superior bacterial clearance of Pseudomonas aeruginosa and improved survival post-infection, with no adverse effects on locomotion, courtship, or sleep architecture. However, under stress paradigms--including sleep deprivation and dextran sulfate sodium (DSS)-induced gut injury--t-Def1 exacerbated intestinal barrier dysfunction, as evidenced by elevated Smurf phenotype incidence, highlighting a trade-off between antimicrobial potency and epithelial vulnerability. Our work establishes Drosophila as a powerful platform for dissecting AMP mechanisms and engineering spatially targeted therapies, offering translational insights for pollinator health and human infectious disease management. These results advocate for iterative refinement of membrane-anchoring strategies to balance therapeutic efficacy with host safety, advancing the development of next-generation AMPs with minimized off-target effects.

biochemistry↗