Biology subjects

Ha, K.

Publications and source records attributed to Ha, K..

13 recordsLinked to original sources

bioRxiv2026

High-throughput discovery of arginine-depleted peptides enables effective antisense delivery for Duchenne muscular dystrophy

Phosphorodiamidate morpholino oligomers (PMOs) are approved exon-skipping antisense therapeutics for Duchenne muscular dystrophy (DMD), but their clinical utility is limited by poor uptake in muscle tissue, necessitating frequent high-dose administration. Cell-penetrating peptides (CPPs) can enhance intracellular delivery of PMOs, yet conventional arginine-rich CPPs often cause dose-limiting toxicity, including renal damage, which hinders their clinical translation. To address this challenge, we developed a high-throughput, charge-based chromatographic enrichment platform capable of screening over 15,000 synthetic peptides, including sequences with noncanonical (abiotic) amino acids. This approach enabled de novo discovery of arginine-depleted CPPs with improved delivery profiles. Four lead candidates demonstrated efficient nuclear PMO delivery with ~10-fold lower in vitro toxicity compared to standard CPPs such as penetratin. The top-performing peptide, CXP1, showed robust splice-switching activity and favorable tolerability in both cellular and animal models. In dystrophic mdx mice, CXP1-PMO conjugates achieved greater exon skipping compared to PMOs conjugated to R6G at equivalent doses. Tissue levels of CXP1-PMO correlated with exon-skipping efficacy, establishing a clear pharmacokinetic-pharmacodynamic relationship. These findings highlight a mechanistically novel and translationally relevant discovery strategy, demonstrating the potential of high-throughput platforms to generate more effective CPP-based delivery vehicles for antisense therapeutics in DMD and related neuromuscular disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/730741v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@84b7f2org.highwire.dtl.DTLVardef@1487850org.highwire.dtl.DTLVardef@d2e263org.highwire.dtl.DTLVardef@10ce371_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Structure-guided deimmunisation of Fel d 1 suppresses allergic effector functions ex vivo

Allergic responses to the major cat allergen Fel d 1 are primarily driven by immunoglobulin E (IgE) cross-linking on effector cells. While conventional allergen-specific immunotherapies often rely on whole-allergen extracts with inherent risks of systemic reactions, rationally engineered variants offer a safer path to desensitisation. We present a structure-guided strategy to deimmunise Fel d 1 by selectively disrupting crucial IgE-binding interfaces. Using a computational pipeline integrating structural mapping and immunoinformatics, we designed 30 single-point mutations targeting immunodominant regions. Ex vivo functional evaluation using sera from cat-allergic individuals demonstrated that specific variants, notably K29G in Chain 1, exhibited significantly diminished IgE reactivity and profoundly suppressed basophil activation. Furthermore, CRISPR/Cas9-mediated introduction of the K29G mutation into feline fibroblasts confirmed that the substitution preserves fundamental cellular proliferation. This work demonstrates that targeted computational design can not only yield superior candidates for safer immunotherapeutics but also establish a viable foundation for generating genetically edited, hypoallergenic cats.

immunology↗

bioRxiv2025

Tropomyosin 1 promotes platelet adhesion and clot contraction separate from its roles in developmental hematopoiesis

Genome-wide associations studies (GWAS) have linked the Tropomyosin 1 (Tpm1) gene locus to quantitative blood trait variation, but related mechanisms are unclear. Tpm1 encodes an actin-binding protein that stabilizes actin filaments and influences cell adhesion, signaling, and actomyosin contractility. Murine Tpm1 deficiency enhances embryonic hemogenic endothelial cell specification, but it was unclear if these effects extended to postnatal hematopoiesis. We used Cdh5Cre or VavCre models to conditionally ablate Tpm1 in endothelium or hematopoietic cells. Both models produced knockout mice in normal Mendelian ratios with complete Tpm1 ablation in postnatal blood. Endothelial Tpm1 deletion increased hemogenic endothelial cell specification, but did not change hematopoietic progenitor cell production nor adult blood counts. This suggested separate roles for Tpm1 in the embryonic and adult blood systems. GWAS suggested genetic architecture specifically linking decreased TPM1 expression to increased platelet count. We examined platelet lifespan and function to explain these findings. Tpm1KO increased platelet lifespan and diminished adhesion to fibronectin and fibrinogen. Decreased platelet clearance could explain increased platelet count in GWAS. Platelet fibrin binding is necessary for blood clot contraction, which reduces vascular occlusion following initial hemostasis. Tpm1KO reduced clot contraction and enhanced clot formation with worsened vascular occlusion in a ferric chloride-induced stroke model. These findings reveal a new role for Tpm1 in platelet function, offering insight into how cytoskeletal regulation impacts human platelet traits and pointing to novel targets to modify stroke risk and thrombotic disease.

cell biology↗

bioRxiv2024

Structure-Based Design of Small-Molecule Inhibitors of Human Interleukin-6

Human Interleukin-6 (hIL-6) is a pro inflammatory cytokine that binds to its receptor, IL-6R followed by binding to gp130 and subsequent dimerization to form a hexamer signaling complex. A critical inflammation mediator, hIL-6 is associated with a diverse range of diseases and monoclonal antibodies are in clinical use that either target IL-6R or hIL-6 to inhibit signaling. Here, we perform high throughput structure-based computational screening using ensemble docking for small molecule antagonists for which the target conformations were taken from 600 ns long molecular dynamics simulations of the apo protein. Prior knowledge of the contact sites from binary complex studies and experimental work was incorporated into the docking studies. The top 20 scored ligands from the in silico studies after post analysis were subjected to in vitro functional assays. Among these compounds, the ligand with second-highest calculated binding affinity showed experimentally [~]84% inhibitory effect on IL6-induced STAT3 reporter activity at 10-5 molar concentration. This finding may pave the way for designing small molecule inhibitors of hIL-6 of therapeutic significance.

bioinformatics↗

bioRxiv2023

Fungi and Bacteria Occupy Distinct Spatial Niches within Carious Dentin

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n=43) and 4 stages of caries progression from children with severe early childhood caries (n=32). Additionally, healthy (n=10) and carious (n=10) primary teeth were decalcified, sectioned, and stained with Grocotts methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal {beta}-D-glucan, gram-positive bacterial lipoteichoic acid, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocotts silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

microbiology↗

bioRxiv2023

ADPKD-Causing Missense Variants in Polycystin-1 Disrupt Cell Surface Localization or Polycystin Channel Function

Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of this monogenic disorder, our limited mechanistic understanding of ADPKD has hindered therapeutic development. Here, we successfully developed bioassays that functionally classify missense variants in polycystin-1 (PC1). Strikingly, ADPKD pathogenic missense variants cluster into two major categories: 1) those that disrupt polycystin cell surface localization or 2) those that attenuate polycystin ion channel activity. We found that polycystin channels with defective surface localization could be rescued with a small molecule. We propose that small-molecule-based strategies to improve polycystin cell surface localization and channel function will be effective therapies for ADPKD patients.

cell biology↗

bioRxiv2023

Pan-cancer analysis reveals multifaceted roles of retrotransposon-fusion RNAs

Transposon-derived transcripts are abundant in RNA sequences, yet their landscape and function, especially for fusion transcripts derived from unannotated or somatically acquired transposons, remains underexplored. Here, we developed a new bioinformatic tool to detect transposon-fusion transcripts in RNA-sequencing data and performed a pan-cancer analysis of 10,257 cancer samples across 34 cancer types as well as 3,088 normal tissue samples. We identified 52,277 cancer-specific fusions with [~]30 events per cancer and hotspot loci within transposons vulnerable to fusion formation. Exonization of intronic transposons was the most prevalent genic fusions, while somatic L1 insertions constituted a small fraction of cancer-specific fusions. Source L1s and HERVs, but not Alus showed decreased DNA methylation in cancer upon fusion formation. Overall cancer-specific L1 fusions were enriched in tumor suppressors while Alu fusions were enriched in oncogenes, including recurrent Alu fusions in EZH2 predictive of patient survival. We also demonstrated that transposon-derived peptides triggered CD8+ T-cell activation to the extent comparable to EBV viruses. Our findings reveal distinct epigenetic and tumorigenic mechanisms underlying transposon fusions across different families and highlight transposons as novel therapeutic targets and the source of potent neoantigens.

cancer biology↗

bioRxiv2023

Assessing the Predictive Ability of Computational Epitope Prediction Methods on Fel d 1 Allergen

While computational epitope prediction methods have found broad application, their use, specifically in allergy-related contexts, remains relatively less explored. This study benchmarks several publicly available epitope prediction tools, focusing on the allergenic IgE and T-cell epitopes of Fel d 1, an extensively studied allergen. Using a variety of tools accessible via the Immune Epitope Database (IEDB) and other resources, we evaluate their ability to identify the known linear IgE and T-cell epitopes of Fel d 1. Our results show a limited effectiveness for B-cell epitope prediction methods, with most performing only marginally better than random selection. We also explored the general predictive abilities on other allergens, and the results were largely random. When predicting T-cell epitopes, ProPred successfully identified all known Fel d 1 T-cell epitopes, whereas the IEDB approach missed two known epitopes and demonstrated a tendency to over-predict. However, when applied to a larger test set, both methods performed only slightly better than random selection. Our findings show the limitations of current computational epitope prediction methods in accurately identifying allergenic epitopes, emphasizing the need for methodological advancements in allergen research.

bioinformatics↗

bioRxiv2022

COOBoostR: an extreme gradient boosting-based tool for robust tissue or cell-of-origin prediction of tumors

We here present COOBoostR (https://github.com/SWJ9385/COOBoostR), a computational method designed for the putative prediction of tissue-or cell-of-origin of various cancer types. COOBoostR leverages regional somatic mutation density information and chromatin mark features to be applied to an extreme gradient boosting-based machine-learning algorithm. COOBoostR ranks chromatin marks from various tissue and cell types which best explain the somatic mutation density landscape of any sample of interest. Through integrating either ChIP-seq based chromatin data or bulk/single cell chromatin accessibility data along with regional somatic mutation density data derived from normal cells/tissue, precancerous lesions, and cancer types, we show that COOBoostR outperforms existing random forest-based methods in prediction speed with comparable or better tissue or cell-of-origin prediction performance. In addition, our results suggest a dynamic somatic mutation accumulation at the normal tissue or cell stage which could be intertwined with the changes in open chromatin marks and enhancer sites. These results further represent chromatin marks shaping the somatic mutation landscape at the early stage of mutation accumulation, possibly even before the initiation of precancerous lesions or neoplasia.

bioinformatics↗

bioRxiv2022

The cilia enriched oxysterol 7β,27-DHC is required for polycystin activation

PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7{beta},27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7{beta},27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.

physiology↗

bioRxiv2020

Monodisperse drops templated by 3D-structured microparticles

The ability to create uniform sub-nanoliter compartments using microfluidic control has enabled new approaches for analysis of single cells and molecules. However, specialized instruments or expertise have been required, slowing the adoption of these cutting-edge applications. Here, we show that 3D-structured microparticles with sculpted surface chemistries template uniformly-sized aqueous drops when simply mixed with two immiscible fluid phases. In contrast to traditional emulsions, particle-templated drops of a controlled volume occupy a minimum in the interfacial energy of the system, such that a stable monodisperse state results with simple and reproducible formation conditions. We describe techniques to manufacture microscale drop-carrier particles and show that emulsions created with these particles prevent molecular exchange, concentrating reactions within the drops, laying a foundation for sensitive compartmentalized assays with minimal instrumentation.

bioengineering↗

bioRxiv2019

Tumor mutational landscape is a record of the pre-malignant state

Chromatin structure has a major influence on the cell-specific density of somatic mutations along the cancer genome. Here, we present a pan-cancer study in which we searched for the putative cancer cell-of-origin of 2,550 whole genomes, representing 32 cancer types by matching their mutational landscape to the regional patterns of chromatin modifications ascertained in 104 normal tissue types. We found that, in almost all cancer types, the cell-of-origin can be predicted solely from their DNA sequences. Our analysis validated the hypothesis that high-grade serous ovarian cancer originates in the fallopian tube and identified distinct origins of breast cancer subtypes. We also demonstrated that the technique is equally capable of identifying the cell-of-origin for a series of 2,044 metastatic samples from 22 of the tumor types available as primaries. Moreover, cancer drivers, whether inherited or acquired, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings highlight that many somatic mutations accumulate while the chromatin structure of the cell-of-origin is maintained and that this historical record, captured in the DNA, can be used to identify the often elusive cancer cell-of-origin.

genomics↗

bioRxiv2019

Somatic mutation landscape reveals differential variability of cell-of-origin for primary liver cancer

BackgroundPrimary liver tissue cancers display consistent increase in global disease burden and mortality. Identification of cell-of-origins for primary liver cancers would be a necessity to expand options for designing relevant therapeutics and preventive medicine for these cancer types. Previous reports on cell-of-origin for primary liver cancers was mainly from animal studies, and integrative research utilizing human specimen data was poorly established.\n\nMethodsWe analyzed a whole-genome sequencing data set for a total of 363 tumor and progenitor tissues along with 423 normal tissue epigenomic marks to predict the cell-of-origin for primary liver cancer subtypes.\n\nResultsDespite the mixed histological features, the predicted cell-of-origin for mixed hepatocellular carcinoma / intrahepatic cholangiocarcinoma were uniformly predicted as a hepatocytic origin. Individual sample-level prediction revealed differential level of cell-of-origin heterogeneity depending on the primary liver cancer types, with more heterogeneity observed in intrahepatic cholangiocarcinomas. Additional analyses on the whole genome sequencing data of hepatic progenitor cells suggest these progenitor cells might not a direct cell-of-origin for liver cancers.\n\nConclusionsThese results provide novel insights on the heterogeneous nature and potential contributors of cell-of-origin predictions for primary liver cancers.

genomics↗