Biology subjects

Kang, Y.

Publications and source records attributed to Kang, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

TDP-43 amyloid conversion in cells tracks the net charge of a polyampholyte motif, not condensate solidification

Liquid-liquid phase separation of TDP-43 is implicated in ALS and FTLD, yet how its condensates convert into pathological amyloid in living cells remains poorly defined. Here we identify a polyampholyte motif (PAM) in the linker between RRM2 and the amyloid-forming low-complexity domain as a charge-tunable determinant of fibrillization that operates independently of condensate solidification. In budding yeast expressing GFP-TDP-43{Delta}NLS, charge reversal of three arginines (R268E/R272E/R275E; 3R-E) converted liquid puncta into ProteoStat-positive fibrils. Unexpectedly, fluorescence recovery after photobleaching showed 3R-E condensates to be more mobile than those of wild type, whereas the least mobile condensates, in yeast and in human U2OS cells, remained ProteoStat-negative. Across 24 PAM charge variants, the proportion of fibril-bearing cells scaled linearly with net charge regardless of residue identity, with charge neutralization delaying rather than abolishing conversion. Phosphomimetic substitution of the PAM serines alone induced fibrils, implicating phosphorylation as a potential physiological modulator of PAM charge.

biochemistry↗

bioRxiv2026

Single-cell DNA cytometry with magnetic- and fluorescence-activated bead sorting

Many clinically important cell populations are defined by intracellular DNA or RNA signatures, but isolating and profiling these cells remains difficult. Probe-based in situ hybridization strategies that are compatible with cytometry have difficulty resolving low-abundance transcripts, integrated provirus, or single-copy genomic mutations. In contrast, droplet digital PCR achieves single-molecule sensitivity; however, subsequent cell isolation requires specialized microfluidic sorters that are slow and not widely available. Here, we introduce magniFIND-seq, a nucleic acid cytometry platform that combines the sensitivity of digital PCR with commercial magnetic-activated (MACS) and fluorescence-activated (FACS) sorting instruments. Single-cell genomes and transcriptomes are captured in agarose beads, target sequences are detected by digital PCR, and bead-bound amplicons are labeled with magnetic or fluorescent probes. Controlled evaporation shrinks beads from 55 to 20 m, enabling scalable magnetic separation, fast single-bead FACS sorting, and their combination for high-purity recovery of rare populations. Using magniFIND-seq, we demonstrate multiplexed single-copy detection and FACS-based isolation of simian immunodeficiency virus proviral pol and env targets. Separately, using BCR::ABL1 as a disease-defining target, we enrich chronic myeloid leukemia cells and recover single-cell transcriptomes that resolve tyrosine kinase inhibitor-resistance programs. magniFIND-seq extends the throughput and accessibility of nucleic acid cytometry by engineering compatibility with commercial instruments.

bioengineering↗

bioRxiv2026

LucaCell: a sequence-centric foundation model for cross-species single-cell analysis

Single-cell foundation models have transformed transcriptomic analysis, yet most rely on fixed gene identifiers that limit transfer across species and data types. Here we present LucaCell, a sequence-centric foundation model that represents genes through pre-trained mRNA sequence embeddings rather than static gene annotations. Gene expression is discretized into bins and modeled with a Transformer encoder, enabling sequence-informed cell representation without a fixed gene-ID vocabulary. Pre-training on 85 million human and mouse single cells, LucaCell is evaluated on human, mouse and lemur gene expression profiles, human chromatin accessibility data, unaligned reads from more than 50 prokaryotic taxa, and five influenza A virus genomes. LucaCell enables manual-mapping-free cross-species cell type annotation and an alignment-free microbial embedding framework that simultaneously distinguishes bacterial species identity and intra-species physiological states. It also improves gene expression reconstruction by incorporating donor-specific exonic SNP information into mRNA sequence embeddings, and predicts cellular viral load across influenza A virus strains while highlighting infection-like transcriptional states in mock-infected cells. These results show that sequence-informed gene representation can improve the generalization of single-cell foundation models across species, data types, and predictive tasks.

bioinformatics↗

bioRxiv2026

A T2T Benchmark Reveals How Reference Choice Shapes Human Genome Interpretation

The completion of telomere-to-telomere (T2T) human genomes has expanded the accessible landscape of human genetic variation, yet benchmark resources remain limited to conventional high-confidence regions defined by existing reference frameworks. Here, we generated a near-perfect diploid T2T genome (T2T-LIN) from a Chinese individual and established assembly-based truth sets by comparison with T2T-YAO, an ancestry-matched near-perfect T2T reference genome. The benchmark showed a heterozygous/homozygous SNV ratio of ~2, consistent with expectations under Hardy-Weinberg equilibrium, and enabled genome-wide evaluation of reference-dependent biases. We found that reference choice substantially influences genome interpretation: ancestry-matched linear T2T references provided the most faithful representation of individual genomic variation and enabled more accurate genome reconstruction than unmatched linear, diploid and graph-based references. Benchmarking previously inaccessible repetitive and structurally complex regions revealed substantial limitations of current variant callers that were masked by conventional metrics. The T2T-LIN and YAO-LIN benchmarks establish a T2T-era framework for evaluating reference-dependent genome interpretation and variant discovery across nearly the complete human genome.

genomics↗

bioRxiv2026

Novel Dissymmetric Ionizable Lipid-Assembled Lipid Nanoparticles for Delivery of Ferroptosis-Related siRNA in Diabetic Treatment

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

pharmacology and toxicology↗

bioRxiv2026

BBBP_Atlas: Unified Interpretable Modeling of Blood Brain Barrier Permeability across Small Molecules and Peptides

Accurate prediction of blood-brain barrier permeability (BBBP) is essential for central nervous system drug discovery, yet existing models are often limited by their reliance on predefined physicochemical descriptors, small-molecule-centered training sets, or conformation-dependent representations, which restricts their transferability across chemically diverse modalities especially peptides. In addition, publicly available BBBP datasets remain fragmented, inconsistently standardized, and weakly controlled for molecular redundancy, increasing the risk of data leakage and overestimated model performance. In this study, we propose BBBP-Atlas, a structure-aware BBB permeability prediction model designed for unified modeling of small molecules and peptides with the first cross-modal dataset OmniBBBP. Designed to bypass descriptor and conformation dependencies, our model represents standardized molecular structures as atom-level graphs to capture local atom-bond environments and long-range topological dependencies associated with BBB transport. This design enables direct learning of structure-permeability relationships from molecular topology. For model training and evaluation, we curated a cross-modal, redundancy-filtered database OmniBBBP that seamlessly unifies small molecules and complex peptides, containing 10,218 unique compounds with 9,316 small molecules and 902 peptides. BBBP-Atlas achieved an accuracy of 0.8914 and an MCC of 0.7678 on the independent test set. On a balanced external benchmark of 200 compounds, our model reached an AUC of 0.9108, an accuracy of 0.8500, and an MCC of 0.7000, outperforming LightBBB by an absolute MCC gain of 6%. Case studies further showed that BBBP-Atlas captured clinically meaningful BBB permeability patterns, correctly identifying lorlatinib as BBB-permeable and vancomycin as BBB-impermeable with high confidence. The OmniBBBP-backed BBBP-Atlas offers a versatile and cross-modal approach for single-compound prediction, batch screening, and dataset exploration for CNS drug discovery. BBBP-Atlas is available at https://cadd.drugflow.com/bbbp/.

bioinformatics↗

bioRxiv2026

Metastatic Osteosarcoma is Characterized by Loss of Osteoblastic Lineage Fidelity

Osteosarcoma (OS) is a rare and aggressive bone cancer with limited therapeutic progress in several decades. To characterize the transcriptional diversity of malignant cell states in OS, we analyzed single-nucleus RNA sequencing data from 24 tumors, including both primary and metastatic lesions. We identified a canonical osteoblastic cell state that predominated in primary tumor cells, but was dampened in metastases. Integrating tumor data with data from embryonic skeletal development and an experimental osteoblast differentiation model revealed that metastatic OS cells resemble poorly specified mesenchyme enriched for non-osseous programs. Clonal populations in metastatic samples acquired mesenchymal cell states distinct from paired primary tumors. Similar cell state shifts were evident in post-treatment primary tumors relative to paired pre-treatment samples. Our results suggest that localized post-treatment and metastatic OS malignant cells lose osteoblastic lineage fidelity, which may represent a unifying axis of disease evolution and reveal new therapeutic vulnerabilities.

Cancer Biology↗

bioRxiv2026

The engulfment receptor CED-1/MEGF10 activates the GPCR LAT-1/ADGRL3 for apoptotic cell degradation

Efferocytosis, the clearance of apoptotic cells, is crucial for tissue homeostasis, inflammation suppression, and repair. While several G protein-coupled receptors (GPCRs) are involved, the broader role of GPCRs in efferocytosis remains unexplored. Through a genome-wide RNAi screen in Caenorhabditis elegans, we identified the adhesion GPCR LAT-1 as a key regulator of apoptotic cell degradation through the promotion of phagosome maturation. Our secondary RNAi scrween for transcriptional regulators revealed that the transcription factor AST-1 acts downstream of LAT-1 to mediate apoptotic cell degradation. We show that the engulfment receptor CED-1 functions as a ligand for LAT-1, activating a Gs protein/adenylyl cyclase/PKA/AST-1 signaling cascade and inducing the transcription of vps-34 and piki-1, which encode phosphatidylinositol 3-kinases essential for PtdIns3P generation on phagosomes. This pathway maintains appropriate VPS-34 and PIKI-1 levels for efficient efferocytosis. Notably, LAT-1 is evolutionarily conserved, with homologs in Drosophila (CIRL) and mammals (ADGRL3) performing similar functions. These findings reveal a conserved CED-1/MEGF10-LAT-1/ADGRL3 axis that orchestrates apoptotic cell clearance via a novel transcriptional regulatory pathway, providing new insight into the molecular mechanisms underlying tissue homeostasis and diseases associated with defective efferocytosis, including autoimmunity and neurodegeneration.

cell biology↗

bioRxiv2026

Co-expression of IBDV VP2 and H9N2 HA by recombinant HVT induces high protection against both pathogens in chickens

Infectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are significant global threats to poultry health and production. While IBDV induces severe immunosuppression, undermining host defense and vaccine efficacy, H9N2 AIV is characterized by widespread prevalence, persistent shedding, and substantial economic losses. Conventional inactivated vaccines often fail to elicit robust cellular immunity and necessitate multiple booster doses, underscoring the urgent requirement for advanced multivalent vaccination platforms. To address this, we developed a recombinant herpesvirus of turkey (rHVT BAC-VP2-HA) using a bacterial artificial chromosome (BAC) vector system, engineered to co-express the major protective antigen VP2 of IBDV and the hemagglutinin (HA) of H9N2 AIV. Genetic stability and in vitro characterization confirmed that the recombinant exhibited replication kinetics and plaque morphology comparable to parental HVT, with stable antigen expression. In SPF chickens, rHVT BAC-VP2-HA induced strong humoral immune responses against both target antigens, comparable to those elicited by a commercial inactivated vaccine. Crucially, the recombinant virus significantly enhanced cellular immunity, evidenced by markedly elevated CD3+CD8+ T cell responses. Upon challenge, the recombinant conferred high clinical protection (86%) against virulent IBDV, significantly ameliorating bursal pathology and reducing viral loads. Notably, it provided complete (100%) protection against H9N2 AIV, effectively abolishing viral shedding and suppressing viral replication in respiratory tissues. These results demonstrate that rHVT BAC-VP2-HA is a safe and efficacious candidate capable of eliciting humoral and cellular immune responses, offering a promising strategy for the integrated control of major poultry diseases. ImportanceInfectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are major pathogens that frequently co-circulate in poultry, where IBDV-induced immunosuppression compromises the efficacy of vaccination against other infectious diseases. Conventional inactivated vaccines primarily induce humoral immunity and are often insufficient to prevent viral shedding or provide broad protection against multiple pathogens. In this study, we developed a recombinant herpesvirus of turkeys (HVT) vaccine co-expressing the IBDV VP2 and H9N2 HA antigens and demonstrated that it induces both robust antibody responses and enhanced CD8+ T cell immunity. Notably, this vaccine not only provided effective protection against IBDV but also completely prevented viral shedding following H9N2 challenge. These findings highlight the advantage of HVT-vectored multivalent vaccines in eliciting balanced immune responses and controlling virus transmission, providing important insights for the development of next-generation vaccines against immunosuppressive and respiratory viral co-infections in poultry.

microbiology↗

bioRxiv2026

Ribosome biogenesis bottlenecks reveal vulnerabilities in cancer

Cell growth requires elevated protein synthesis, which depends on the production of ribosomes. Ribosome biogenesis is a complex, multi-step pathway in which newly transcribed precursor ribosomal RNA (rRNA) undergoes coordinated processing and assembly in the nucleolus to produce the small and large ribosomal subunits (SSU and LSU).1-3 Oncogene activation stimulates rRNA transcription and processing, giving rise to enlarged nucleoli that produce thousands of ribosomes every minute.4,5 However, efficient ribosome production requires tight coordination across numerous maturation steps, and it remains unclear if elevated rDNA transcription is proportionally converted into mature ribosomes, or whether imperfect coordination constrains the output yield. Here, we quantify pre-rRNA transcription (input) and compare it with newly-assembled cytoplasmic ribosomes (output), revealing that oncogene activation reduces the efficiency of ribosome production. Using a quantitative pulse-chase sequencing approach with mathematical modeling to resolve rRNA maturation kinetics, we found that oncogene activation creates late-stage processing bottlenecks, characterized by delayed precursor maturation and increased degradation. Perturbation of late-stage ribosome biogenesis factors preferentially impaired oncogene-driven cell growth, and limited tumor growth in mouse models, suggesting that these bottlenecks represent selective vulnerabilities in cancer, created by imbalanced biosynthetic flux. Together, these findings reveal that oncogene-driven ribosome production is imperfectly coordinated across maturation steps, and suggest that capacity limits in multi-step assembly pathways may be therapeutically exploitable in cancer and other diseases.

cell biology↗

bioRxiv2026

Metabolic compensation via gluconeogenesis explains the non-essentiality of glycogen phosphorylase as an insecticidal target in Plutella xylostella

Glycogen phosphorylase (GP) catalyzes the rate-limiting step of glycogenolysis and occupies a central position in insect carbohydrate metabolism, supplying precursors for chitin biosynthesis. Acyl urea compounds structurally related to benzoylphenylurea (BPU) insecticides are potent inhibitors of mammalian GP, raising the question of whether insect GP could serve as an independent insecticidal target. Here, we systematically evaluate this possibility in the diamondback moth Plutella xylostella. We show that a mammalian GP inhibitor (GPI) potently inhibits recombinant PxGP (IC50 = 2.96 nM), while the BPU insecticide diflubenzuron (DFB) does not. Molecular docking and MM/GBSA analysis predict that this selectivity reflects differential side-chain engagement: GPI is predicted to occupy the allosteric site at the dimer interface via contacts with seven residues spanning both subunits ({Delta}G = -34.63 kcal/mol), whereas DFB's difluorobenzoyl moiety is predicted to remain solvent exposed without productive protein contacts ({Delta}G = -29.29 kcal/mol). Despite potent in vitro inhibition, GPI exhibits no insecticidal activity, and RNAi-mediated knockdown of PxGP (confirmed by enzyme activity measurements showing ~29% reduction in per-larva GP-a activity) does not impair development or survival. We provide evidence that insects compensate through a multi-layered metabolic response: upregulation of gluconeogenic enzymes (PEPCK, G-6-Pase), selective upregulation of glycogen branching enzyme (GBE) but not -amylase, and changes in protein content suggestive of catabolic substrate mobilization. Fitness assessment reveals transient larval weight loss (24-48 h) with complete recovery of pupal weight, adult wing morphology, and female fecundity, indicating that the metabolic compensation response is ultimately effective in sustaining development. These findings suggest that GP is functionally non-essential under these conditions, likely through a combination of gluconeogenic compensation and the availability of alternative carbon sources, and highlight the importance of considering metabolic network redundancy in target-based insecticide design.

biochemistry↗

bioRxiv2026

Ultrasound transcutaneous auricular vagus nerve stimulation enhances semantic processing

Efficient access to conceptual knowledge is central to everyday cognition, yet the effects of non-invasive neuromodulation on semantic retrieval remain insufficiently characterised. Transcutaneous auricular vagus nerve stimulation (taVNS) offers a mechanistically distinct route to modulate distributed cognitive systems via ascending vagal afferent pathways, but its effects on semantic processing has not been explored. Here, we tested whether ultrasound-based taVNS delivered to the right cymba conchae can acutely enhance semantic retrieval efficiency in healthy adults. Twenty-seven participants completed a single blind, sham-controlled study comprising two counterbalanced sessions (active vs. sham) in a within-subject design, separated by at least five days. In each session, participants performed a semantic association task and a number-judgement control task before and immediately after 30 minutes of stimulation. Active taVNS produced selective post-stimulation improvements in semantic retrieval performance relative to sham and baseline, while performance on the control task remained unchanged. Adverse effects were minimal and did not differ between active and sham conditions. These findings provide causal evidence that ultrasound-based taVNS can acutely improve the efficiency of semantic processing, expanding the cognitive profile of taVNS beyond predominantly episodic and emotional memory domains and supporting its feasibility as a scalable approach for memory enhancement.

neuroscience↗

bioRxiv2025

LISA: A Comprehensive R Package for Lentiviral Integration Site Analysis in Gene Therapy Safety Assessment

BackgroundGene therapy using integrating viral vectors has shown remarkable clinical success, but the random integration of vectors into the host genome poses potential safety concerns, particularly insertional mutagenesis. Common Integration Sites (CIS) near oncogenes significantly increase the risk of clonal expansion and malignant transformation. Current tools for integration site (IS) analysis lack comprehensive functionality for safety assessment, particularly in identifying risk-associated genomic regions and tracking clonal dynamics over time. ResultsWe developed LISA (Longitudinal Integration Site Analysis), a comprehensive R package specifically designed for gene therapy safety evaluation. LISA incorporates a novel approach that trains integration site profiles using cumulative curves derived from normal cell infusion samples and normal patient samples, with subsequent statistical testing to distinguish these normal samples from those with exceptional clonal expansion, thereby enabling effective identification of dominant clone formation. For longitudinal studies, LISA monitors fluctuations in clonal dynamics via Richness and Evenness metrics, and integrates this cumulative curve-based statistical testing approach into its early warning system to detect potentially dominant clones. Additionally, LISA features an optimized CIS detection algorithm with O (n log n) time complexity, achieved through chromosome-position encoding and a seed-site strategy, which facilitates efficient analysis of large-scale clinical datasets. The package also provides systematic functional annotations, including annotations for Safe Harbor regions, enhancer/promoter elements, and proximity to three curated gene sets: adverse event-associated genes (AE genes, n=36), cancer-related genes (n=980), and immune-related genes (n=2,485). Furthermore, it encompasses six categories of interactive visualizations and has been validated using real clinical data from gene therapy trials.

bioinformatics↗

bioRxiv2025

Neat1 alleviates ischemic injury in skeletal muscles

Peripheral artery disease (PAD) has historically been regarded as an age-related vascular disorder; however, recent attention has shifted toward the myopathic components of the disease. Conventional interventions, such as revascularization, have had limited success in reversing muscle pathology or preventing adverse outcomes like amputation. Hypoxia-inducible factors (HIFs) are key regulators of cellular responses to ischemia, including the promotion of angiogenesis and glycolysis. While pharmacological stabilization of HIF proteins represents a promising therapeutic strategy, its efficacy is diminished in aged muscles due to their intrinsically low basal HIF expression. We hypothesized that long noncoding RNAs (lncRNAs) might enhance the hypoxic response in aged muscle through post-transcriptional regulation of HIF expression. Our study identified a long noncoding RNA Neat1, as a critical mediator of the hypoxia-induced stress response, including upregulation of Hif1. In a murine hindlimb ischemia model, Neat1 knockout mice exhibited extensive necrosis following femoral artery ligation, whereas Neat1 overexpression conferred protection against ischemic injury. Mechanistically, we found that Neat1 regulates the stability of Hif1a mRNA, as Hif1a transcript levels were significantly reduced in Neat1-deficient muscle cells. Importantly, aged muscles displayed a blunted hypoxic response due to diminished Hif1a expression--an effect that was reversed through Neat1 overexpression, resulting in improved resistance to ischemic damage. In summary, our findings highlight Neat1 as a novel regulator of muscle adaptation to hypoxia in aging. Enhancing Neat1 expression may represent a promising therapeutic strategy for improving ischemic outcomes in patients with peripheral artery disease.

cell biology↗

bioRxiv2025

Argonautes and small RNAs associated with nematode programmed DNA elimination

Programmed DNA elimination (PDE) selectively removes DNA in pre-somatic cells to form the somatic genome during development. Our mechanistic understanding of PDE is mainly based on studies in the single-cell ciliates, where small RNAs and transposases are involved. In contrast, the mechanism(s) of metazoan PDE remains largely unknown. In the parasitic nematode Ascaris, PDE removes and remodels all germline chromosome ends and partitions fused germline chromosomes into somatic chromosomes, resulting in dramatic karyotype changes. Here, we identified two Ascaris Argonautes, AsWAGO-2 and AsWAGO-3, associated with retained or eliminated chromosome regions, respectively, during PDE mitoses. AsWAGO-3 siRNAs target primarily genes. However, a subset of AsWAGO-3 siRNAs targets repeats within the eliminated regions and located in the middle of chromosomes. These chromosome regions are derived from the fusion of the ends of ancestral chromosomes. In contrast, AsWAGO-2 and its associated siRNAs are transiently enriched on retained chromosomes during elimination mitoses. Overall, our data reveal a link between Ascaris WAGOs, their siRNAs, and PDE, identifying small RNA pathways that selectively distinguish retained and eliminated DNA. We suggest that AsWAGO-3 associated small RNAs targeting the interior of select chromosomes may be a response to the fusions of germline chromosomes.

molecular biology↗

bioRxiv2025

A complete diploid human genome benchmark for personalized genomics

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2-7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.

genomics↗

bioRxiv2025

Polyelectrolyte domains in intrinsically disordered regions differentially regulate intracellular biomolecular condensation

Intracellular condensation is a hallmark of many proteins containing intrinsically disordered regions (IDRs). Decoding the regulatory mechanisms of condensation encoded within the amino acid sequences of condensate-forming proteins is essential for understanding both how intracellular condensates assemble and how they contribute to various disease pathologies. Here, we investigated the budding yeast P-body proteins Nst1 and Edc3, which exhibit pronounced intracellular self-condensation, and identified charged amino acid clusters in the low complexity regions (LCRs) that modulate their phase behavior. Guided by the stickers-and-spacers framework, we hypothesized that clusters of charged residues within IDRs function in promoting intracellular condensation process. Using a sliding-window approach, we designated and dissected the polyelectrolyte (PE) domains of Nst1 and Edc3 and established their essential contributions to condensate coalescence. In Nst1, the polyanionic (PA) subdomain enhances intermolecular attraction and promotes coalescence, whereas the polyampholyte (PAM) subdomain imparts rigidity to the condensate as a distinct modulatory element and functions as a platform in condensation process of other P-body components. PA of Edc3 also regulates coalescence. Extending these findings to the neurodegenerative disease-associated protein TDP-43, we show that its PA regions are critical for coalescence, consistent with the presence of negatively charged amino acid clusters as potent molecular stickers functioning in condensate coalescence in cells. These results highlight charged-residue clusters as important sequence determinants of protein phase behavior and suggest that they represent promising novel regulatory targets for modulating TDP-43 condensation in cells.

cell biology↗

bioRxiv2025

Transcriptional Rewiring of BET Inhibitor Treated Ewing Sarcoma Cells Augments their Dependency on Focal Adhesion Kinase

Epigenetic dysregulation is a hallmark of cancer. Small molecule inhibitors such as bromodomain and extraterminal (BET) protein inhibitors developed to target epigenetic dependencies have demonstrated significant promise in preclinical models. However, clinical success with epigenetic drugs as single agents has been limited by emergence of tumor cell tolerance and escape, which often occurs due to transcriptional rewiring. Ewing sarcoma (EwS), a bone and soft tissue tumor driven by the EWS::FLI1 fusion oncoprotein, is characterized by profound oncogene-dependent enhancer reprogramming. Thus, epigenetic modifying therapies are of high therapeutic interest. In this study, we sought to elucidate how EwS cells escape BET inhibition to identify biologically informed drug combinations that could be advanced to clinical trials. As expected, EwS cells and xenografts initially responded to BMS-986158, a pharmaceutical grade BET inhibitor, but proliferation was rapidly restored. A kinase inhibitor screen showed that BMS-986158 drug tolerant persister (DTP) cells were sensitive to inhibitors of Focal Adhesion Kinase (FAK), a critical signaling node downstream of extracellular matrix (ECM) engagement. RNA sequencing revealed that DTP cells had been transcriptionally rewired and that mesenchymal signature and ECM remodeling genes were specifically upregulated. Combining BMS-986158 with the FAK inhibitor Defactinib had synergistic effects, reducing EwS cell proliferation, survival, and invasion in vitro, and significantly inhibited tumor outgrowth in vivo. Our studies identify BET and FAK inhibition as a rational combination therapy worthy of further investigation for EwS, and demonstrate that defining emergent mechanisms of epigenetic drug tolerance can identify new vulnerabilities that can be therapeutically targeted.

cancer biology↗