Biology subjects

Huang, Y.-T.

Publications and source records attributed to Huang, Y.-T..

At least 19 recordsLinked to original sources

bioRxiv2026

Somatic haplotype reconstruction and variant recalibration from tumor-only long-read sequencing

Separating somatic from germline variants and reconstructing somatic haplotypes are the two central problems of tumor-only cancer genome analysis. Long reads carry the linkage needed to solve both, but chromosome-scale loss of heterozygosity (LOH) and an unknown degree of normal-cell admixture blur the distinction between somatic and germline haplotypes. Here we present LongPhase-TO, the first method to reconstruct somatic haplotypes from a tumor sample alone. Rather than mapping somatic variants onto germline haplotypes, LongPhase-TO co-phases germline and somatic alleles in a unified graph, in which LOH and tumor DNA fraction are resolved internally from heterozygosity depletion and haplotype imbalance rather than a copy-number and ploidy model. Across eight datasets from six cancer cell lines, LongPhase-TO increased haplotype block N50 by a median of 2.9-fold relative to germline phasers. It also consistently improved somatic single-nucleotide variant (SNV) and indel calls from ClairS-TO and DeepSomatic-TO, raising mean F1 from 0.55 to 0.62 and 0.65 for SNVs and from 0.19 to 0.23 for indels, with the largest gains at low tumor DNA fraction. Across breast, melanoma and lung cancer cell lines, LongPhase-TO improves the accuracy of existing somatic callers and reconstructs megabase-scale somatic haplotypes.

bioinformatics↗

bioRxiv2026

Molecular and cell type-specific determinants of inferior colliculus development and auditory function

Sound perception requires sensory information processing through brain regions where alterations to cellular composition or function may impact auditory-relevant behaviors. The inferior colliculus (IC) is a central midbrain hub for integrating and transforming auditory information prior to relay to the forebrain. However, the molecular logic that underlies the cell type specification of the IC remains unknown. Here, using a multiomic approach, we define the transcriptional and chromatin landscapes that underlie IC cell type diversity. We identify distinct glutamatergic neuronal subclasses and the gene regulatory programs that drive their specification, maturation, and survival. We show that perturbation to the transcription factor FOXP2, previously implicated in speech and language as well as brain disorders with altered sensory processing, selectively disrupts the specification and survival of three newly defined glutamatergic neuronal subclasses. We link these molecular and cellular disruptions to functional deficits in auditory processing including altered brainstem and forebrain responses and impaired behavioral sensitivity to acoustic stimuli. Together, these results link gene regulatory mechanisms to cell type-specification in the IC, providing insight into how molecular control of neuronal identity in midbrain sensory centers contributes to systems-level processing of sound.

neuroscience↗

bioRxiv2026

Drug Functional Site-Unknown Molecular Targets Exemplified by SARS-CoV-2 pseudoknot and ribosomal frameshifting-modulators

The -1 Programmed Ribosomal Frameshifting (-1 PRF) signal of SARS-CoV-2, driven by a conserved three-stemmed RNA pseudoknot (PK), is indispensable for viral replication and represents a structurally stable yet underexplored therapeutic target. Unlike rapidly mutating viral proteins, this RNA element offers an opportunity for durable intervention but has historically been considered "undruggable". We developed an integrative drug discovery and characterization pipeline that combines molecular docking, molecular dynamics simulations, and dual-luciferase assays to systematically identify and validate frameshifting-efficiency (Feff) modulators from FDA-approved compounds. To move beyond traditional similarity-based screening, we introduced a contact-distribution-matching method, which ranks candidate compounds by comparing their predicted RNA interaction fingerprints with those of reference modulators. This computational approach, paired with experimental validation, enabled us to expand the repertoire of Feff modulators and establish correlations between binding patterns and functional outcomes. To uncover the underlying mechanisms, we applied steered molecular dynamics simulations and single-molecule optical tweezers measurements, revealing that Feff-enhancing modulators preferentially stabilize the remote stem (stem 3) of the PK, promoting variety of intermediate force species with the beginning base pairs of stem 1 being refolded, even after those base pairs have been unwound by ribosome. The refold of the tips of the stem 1 in turn "push back" the ribosome on the slippery sequence to result in enhanced frameshifting. On the other hand, Feff-suppressing modulators rigidify early stem regions, increasing resistance to ribosomal progression and increase the drop-off rate, eventually leading to a reduced -1 frame to 0 frame ratio in translation. Together, these findings provide the first integrated demonstration of how small molecules can modulate -1 PRF by altering RNA PK folding dynamics. More broadly, our framework establishes a generalizable strategy for rationally targeting structured RNAs with repurposed drugs and offers new opportunities to expand the druggable genome to include noncoding RNA elements and other biomolecular targets lacking known functional sites.

biochemistry↗

bioRxiv2025

LongPhase-S: purity estimation and variant recalibration with somatic haplotying for long-read sequencing

Accurate detection of somatic variants is crucial for precision oncology, and long-read sequencing offers unprecedented advantages in resolving complex cancer genomes. However, most long-read somatic callers rely on phasing built for a diploid genome, an assumption violated by various contamination, subclonal heterogeneity, and aneuploidy in tumors. We present LongPhase-S, a novel method that jointly reconstructs somatic haplotypes, infers tumor purity, and recalibrates somatic variants in a purity-aware manner for paired tumor-normal long-read sequencing. By anchoring each somatic read to a parental germline lin-eage, LongPhase-S provides a phase-resolved view in which germline and somatic reads are disentangled across the genome. Building on somatic haplotyping, LongPhase-S trains a phase-aware purity estimator that outperformed existing methods. Using eight benchmark datasets comprising six cancer cell lines, including breast, melanoma, and lung cancers, LongPhase-S boosted the accuracy of state-of-the-art somatic callers wuth the estimated purity and somatic haplotypes. Specifically, mean F1 scores increased by 4.5% and 7.1% for single-nucleotide variants and insertions and deletions with ClairS, and by 1.2% and 0.5% with DeepSomatic. Collectively, these results showed that somatic haplotyping is a critical yet missing piece in existing somatic callers, which enables purity-aware and phase-resolved variant interpretation in heterogneous tumors.

bioinformatics↗

bioRxiv2025

Phylogenetic Insights into Antifungal Susceptibility: Comparing Environmental and Clinical Scedosporium and Lomentospora Isolates from Taiwan

ObjectivesScedosporium and Lomentospora species are intrinsically resistant molds increasingly implicated in invasive infections with poor outcomes. Olorofim, a novel orotomide antifungal, has demonstrated potent activity against various molds. This study evaluated the antifungal susceptibility of clinical and environmental isolates, focusing on olorofim, and examined these data in a phylogenetic context. MethodsA total of 216 isolates (Scedosporium spp., n=205; L. prolificans, n=11) were collected from clinical and environmental sources across Taiwan. Species identification was confirmed by sequencing of the ITS and {beta}-tubulin regions, followed by maximum-likelihood phylogenetic analysis. Antifungal susceptibility was determined using CLSI M38-A3 method. Cross-resistance was evaluated using correlation analyses. Phylogenetic signal, Mantel tests, and variance partitioning were applied to assess the evolutionary structuring of minimum inhibitory concentrations (MICs). ResultsScedosporium apiospermum (46.3%) and S. boydii (15.7%) were the predominant species, exhibiting comparable triazole susceptibility profiles (MIC90: voriconazole 1-2 mg/L, posaconazole 4 mg/L, and isavuconazole 8 mg/L). L. prolificans displayed pan-resistance to all agents except olorofim. Among triazoles, voriconazole yielded the lowest MICs for most Scedosporium isolates. Olorofim exhibited the lowest MICs across Scedosporium spp. and L. prolificans (all MIC90 [≤]0.12 mg/L). Rare taxa (S. marinum, S. haikouense, S. multisporum, S. minutisporum, S. sp. nov.1) were also identified, exhibiting variable triazole MICs but consistently low olorofim MICs. The association between antifungal susceptibility and species relatedness varied by drug: moderate for posaconazole, amphotericin B, and voriconazole; intermediate for olorofim; weak for isavuconazole; and absent for itraconazole. Most MIC variability (72.5-89.2%) occurred within rather than between species. ConclusionsAntifungal susceptibility among Scedosporium spp. and L. prolificans was drug dependent. Olorofim demonstrated consistently low MICs across species, whereas amphotericin B and triazoles showed variable, species-specific activity. These findings highlight olorofim as a promising therapeutic option and emphasize the need for target gene-based investigations to elucidate the evolutionary basis of antifungal resistance.

microbiology↗

bioRxiv2025

Gliomas phenocopy an inborn error of metabolism to drive neuronal activity and tumor growth

The metabolic hallmarks of high-grade glioma (HGG) are not fully understood. Human brain tissue metabolomics revealed that the creatine synthesis pathway intermediate guanidinoacetate (GAA) accumulated [~]100-fold in HGGs relative to controls, which was caused by imbalanced activities of enzymes in this pathway. Glioma cells secreted GAA rather than using it to produce creatine, implicating an unexpected function. GAA accumulates in GAMT deficiency, an inborn error of metabolism, and elevates neuronal excitability. Neuronal excitability is also increased in glioma and drives tumor growth through neuron-glioma interactions. We hypothesized that glioma-generated GAA excites surrounding neurons. Indeed, GAA induced neuronal hyperactivity by activating GABAA receptors and causing depolarizing GABA currents in glioma-associated neurons with dysregulated chloride homeostasis. Depleting tumoral GAA decreased electrochemical activity, neuron-glioma interactions, and tumor aggressiveness. Our findings unveil a new mechanism linking cancer metabolism with cancer neuroscience and leverage human genetics to nominate GAA synthesis as a target in gliomas.

cancer biology↗

bioRxiv2025

A continuum of information-based temporal stability measures and their decomposition across hierarchical levels

When biodiversity-stability relationships are assessed from temporal patterns of species (or species assemblages) biomass or other key variables, ecological stability is commonly quantified as the inverse of the coefficient of variation (CV) or its square. However, just as biodiversity cannot be fully characterized by a single value, the complexity of temporal stability/invariability cannot be completely captured by one metric, especially since CV is disproportionally sensitive to large data values. Ecologists now recognize that species diversity can be fully characterized by a continuum of Hill-number-based measures parametrized by a diversity order q[≥]0, which determines the sensitivity of the measure to species abundance. Building on the intuitive concept that temporal stability can be quantified by the closeness between the data vector and the ideally maximally stable vector, we propose a continuum of information-based measures of temporal stability/invariability parameterized by an order q>0. This continuous parameter q determines the sensitivity of the measure to the magnitude of biomass or other ecosystem functions. By varying q, researchers can differentially weight small, medium, or large values in a time series, thereby disentangling their respective contributions to stability. Our framework unifies and generalizes classical measures: the case q=1 links to Shannon entropy, reflecting MacArthurs 1955 stability concept; q=2 connects to the conventional CV-based measure. Unlike these traditional metrics, our approach explicitly accounts for the number of data values (i.e., time points), adjusting for time-series-length effects to enable fair and meaningful comparisons across datasets of varying lengths. We extend the framework to hierarchical structures by developing additive and multiplicative decompositions of stability in a metacommunity (or metapopulation) into alpha and beta components. The beta component can be further used to obtain measures that quantify (a)synchrony among communities (or populations). For q=2, the resulting (a)synchrony measure provides a mathematically rigorous, CV-based metric. The proposed measures are illustrated using 22-year biomass time series data from the Jena Experiment in Germany. We developed the R package iSTAY (information-based stability measures) and online tools for computation and visualization. Our measures are adaptable to other functions beyond biomass and are applicable in both temporal and spatial contexts. Open Research StatementThe original biomass data of the Jena Experiment, covering the years 2003 to 2023, can be retrieved from https://jexis.idiv.de/ddm/Data/ShowData/624, while the 2024 data are available at https://jexis.idiv.de/ddm/Data/ShowData/695. These datasets, along with the R code used in this study, are currently accessible on Github at https://github.com/AnneChao/MS_iSTAY for review purposes and will be archived on Zenodo upon journal acceptance.

ecology↗

bioRxiv2025

Versatile entomopathogenic activity of Purpureocillium takamizusanense against diverse agricultural pests

Purpureocillium takamizusanense is an emerging entomopathogenic fungus with significant potential for managing diverse agricultural pests sustainably. This study assesses the broad-spectrum pathogenic activity of P. takamizusanense strain TCTeb01, isolated from naturally infected lychee stink bugs (Tessaratoma papillosa) in Taiwan. The fungus was evaluated against three economically critical agricultural pests: the coffee berry borer (Hypothenemus hampei), southern yellow thrip (Thrips palmi), and lychee stink bug. Pathogenicity assays revealed effective control against all tested pests, showing strong pathogenic capabilities and a clear concentration-dependent efficacy. The fungus demonstrated rapid infection and significant pest mortality, highlighting its viability as a biological control agent. Comprehensive morphological and molecular analyses confirmed the fungal strains identity and documented additional morphological features, expanding the known variability within the species. Overall, the findings underscore the versatility and robust pathogenic potential of P. takamizusanense. Its effectiveness against pests from different insect orders makes it a valuable addition to integrated pest management strategies. Given its environmental compatibility and reduced ecological impact compared to chemical pesticides, P. takamizusanense can represent a sustainable alternative for agriculture, contributing positively to pest management practices and ecosystem health.

microbiology↗

bioRxiv2025

Activation of a Src-JNK pathway in unscheduled endocycling cells of the Drosophila wing disc induces a chronic wounding response

The endocycle is a specialized cell cycle during which cells undergo repeated G / S phases to replicate DNA without division, leading to large polyploid cells. The transition from a mitotic cycle to an endocycle can be triggered by various stresses, which results in unscheduled, or induced endocycling cells (iECs). While iECs can be beneficial for wound healing, they can also be detrimental by impairing tissue growth or promoting cancer. However, the regulation of endocycling and its role in tissue growth remain poorly understood. Using the Drosophila wing disc as a model, we previously demonstrated that iEC growth is arrested through a Jun N-Terminal Kinase (JNK)-dependent, reversible senescence-like response. However, it remains unclear how JNK is activated in iECs and how iECs impact overall tissue structure. In this study, we performed a genetic screen and identified the Src42A-Shark-Slpr pathway as an upstream regulator of JNK in iECs, leading to their senescence-like arrest. We found that tissues recognize iECs as wounds, releasing wound-related signals that induce a JNK-dependent developmental delay. Similar to wound closure, this response triggers Src-JNK-mediated actomyosin remodeling, yet iECs persist rather than being eliminated. Our findings suggest that the tissue response to iECs shares key signaling and cytoskeletal regulatory mechanisms with wound healing and dorsal closure, a developmental process during Drosophila embryogenesis. However, because iECs are retained within the tissue, they create a unique system that may serve as a model for studying chronic wounds and tumor progression. Article summaryThe effects of unscheduled endocycles on tissue growth remain unclear. To investigate this, we used Drosophila to induce a switch from the mitotic cycle to the endocycle and analyzed tissue responses at both the signaling and tissue structure levels. Surprisingly, tissues recognized endocycling cells as wounds, activating regeneration signals and remodeling tissue structure. However, because these cells resist apoptosis, they persist within the tissue without being cleared. This persistence disrupts normal healing, revealing the similarities between unscheduled endocycling cells and chronic wounds. Our system has the potential to serve as a novel model for studying chronic wound responses or tumorigenesis.

developmental biology↗

bioRxiv2025

Mifepristone alone and in combination with scAAV9-SMN1 gene therapy improves disease phenotypes in Smn2B/- spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene. SMA is characterised by alpha motor neuron loss in the spinal cord and subsequent muscle atrophy. There are currently three approved SMN-directed therapies for SMA patients. While these therapies have transformed what was once a life-limiting condition into one that can be managed and even improved, they are unfortunately not cures, highlighting the need for additional supporting second-generation therapies. These should not only target the neuromuscular system but also peripheral and metabolic perturbations that are present in both SMA models and patients. Kruppel-like factor 15 (Klf15) is a transcription factor that maintains metabolic homeostasis and is involved in the glucocorticoid-glucocorticoid receptor (GR) signalling pathway, in several peripheral and metabolic tissues in SMA mice. Here, we used murine and human cellular models as well as SMA mice and Caenorhabditis Elegans (C. elegans) to assess the therapeutic potential of reducing Klf15 activity with mifepristone, a glucocorticoid antagonist, combined with SMN-targeted gene therapy. We report that mifepristone reduces Klf15 expression across several in vitro models, ameliorates neuromuscular pathology in SMA smn-1(ok355) C. elegans and improves survival of SMA Smn2B/- mice. Furthermore, we show that combining mifepristone with an approved SMN-directed gene therapy (scAAV9-SMN1) results in improved tissue- and sex-specific responses to treatment. Our study demonstrates that a multi-tissue targeting SMN-independent drug, alone and in combination with an approved SMN-dependent therapy, has the potential to improve SMA disease pathology.

pharmacology and toxicology↗

bioRxiv2024

Human activity, not environmental factors, drives Scedosporium and Lomentospora distribution in Taiwan

Scedosporium and Lomentospora species are emerging fungal pathogens capable of causing severe infections in both immunocompetent and immunocompromised individuals. Previous environmental surveys have suggested potential associations between these fungi and various soil chemical parameters, though the relative influence of human activity versus environmental factors has not been systematically evaluated. Here, we conducted a comprehensive survey of 406 soil samples from 132 locations across Taiwan, analyzing fungal abundance alongside soil physicochemical parameters and the Human Footprint Index (HFI). We recovered 236 fungal isolates comprising 10 species, with S. boydii (32.2%), S. apiospermum (30.9%), and S. dehoogii (14.4%) being the most prevalent. The highest fungal burdens were observed in urban environments (up to 1293 CFU/g), particularly in public spaces and healthcare facilities. Statistical analysis revealed a significant positive correlation between fungal abundance and HFI (r = 0.125, p = 0.013), while soil chemical parameters including nitrogen, carbon, pH, electrical conductivity, and various base cations showed no significant associations despite their wide ranges. These findings indicate that anthropogenic disturbance of environments, rather than soil chemistry, is the primary driver of Scedosporium and Lomentospora distribution in Taiwan. This understanding holds important implications for predicting infection risks and developing targeted public health strategies, particularly in rapidly urbanizing regions. Future studies incorporating more specific indicators of human impact may further elucidate the mechanisms underlying these distribution patterns. Lay AbstractWe found that human activities, rather than soil properties, determine where Scedosporium/Lomentospora fungi distribute in Taiwan. These fungi are more abundant in urban areas than less-human-disturbed environments, suggesting increased infection risks in densely populated regions.

ecology↗

bioRxiv2024

The Taiwan Precision Medicine Initiative: A Cohort for Large-Scale Studies

The Taiwan Precision Medicine Initiative (TPMI), a project initiated by the Academia Sinica in collaboration with 16 major medical centers around Taiwan, has recruited 565,390 participants who consented to provide DNA samples for genetic profiling and grant access to their electronic medical records (EMR) for studies to develop precision medicine. Access to the EMR is both retrospective and prospective, allowing researchers to conduct prospective studies over time. Genetic profiling is done with population-optimized SNP arrays for the Han Chinese populations that enable genetic analyses such as genome-wide association, phenome-wide association, and polygenic risk score studies to evaluate common disease risk and pharmacogenetic response. Furthermore, the TPMI participants agree to be contacted for future research opportunities related to their genetic risks and receive personalized genetic risk profiles with health management recommendations. TPMI has established the TPMI Data Access Platform (TDAP), a central database and analysis platform that both safeguards the security of the data and facilitates academic research. The TPMI is the largest non-European cohort that merges genetic profiles with EMR in the world. With a cohort that can be followed over time, it can be utilized to validate genetic risk prediction models, conduct clinical trials to show the efficacy of risk-based health management, and optimize health policies based on genetic risks. In this report, we describe the TPMI study design, the population and genetic characteristics of the TPMI cohort, and the power it provides to conduct crucial studies in developing precision medicine on a population and personal level. As Han Chinese represent almost 20% of the worlds population, the results of TPMI studies will benefit >1.4 billion people around the world and serve as a model for developing population-based precision medicine.

genetics↗

bioRxiv2024

Genome diversification of symbiotic fungi in beetle-fungus mutualistic symbiosis

Ambrosia beetles and their fungal symbionts represent a widespread and diverse insect-fungus mutualism. This study investigates the genomic adaptations associated with the evolution of the ambrosia lifestyle across multiple fungal lineages. We performed comparative genomic analyses on 70 fungal genomes from four families (Irpicaceae, Ceratocystidaceae, Nectriaceae, and Ophiostomataceae), including 24 ambrosia and 34 non-ambrosia lineages. Our phylogenomic analyses reveal multiple independent colonization of insect vectors by the fungi, spanning from the mid-Cretaceous (114.6 Ma) to the early Quaternary (1.9 Ma). Contrary to expectations for obligate symbionts, ambrosia fungi showed no significant genome-wide reductions in size, gene count, or secreted protein repertoire compared to their non-symbiotic relatives. Instead, we observed conservation of most assessed genomic features; where genome traits differ between free-living relatives and ambrosia fungi, the changes are lineage-specific, not convergent. Key findings include lineage-specific expansions in carbohydrate-active enzyme families (AA4 in Nectriaceae, CE4 in Ophiostomataceae, and GH3 in Ophiostomataceae and Ceratocystidaceae), suggesting potential enhancement or loss of lignin modification, hemicellulose deacetylation, and cellulose degradation in different ambrosia lineages. Repeat-Induced Point mutation analysis revealed family-specific patterns rather than lifestyle-associated differences. These results highlight the diverse genomic strategies employed by ambrosia fungi, demonstrating that symbiont evolution can proceed through refined, lineage-specific changes rather than genome-wide, or convergent alterations. Unlike other insect-associated fungi, ambrosia fungi do not seem to be domesticated crops, but rather free-living fungi which co-opted wood boring beetles as vectors via subtle, lineage-specific adaptations.

evolutionary biology↗

bioRxiv2024

Cost-effective solutions for high-throughput enzymatic DNA methylation sequencing

Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)--which profiles [~]4 million CpG sites--for miniaturization, flexibility, and multispecies use at a cost of [~]$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R{superscript 2} = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R{superscript 2} = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations--the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia--and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.

genomics↗

bioRxiv2024

Syngeneic natural killer cell therapy activates dendritic and T cells in metastatic lungs and effectively treat low-burden metastases

Natural killer (NK) cells can control metastasis through cytotoxicity and IFN-{gamma} production independently of T cells in experimental metastasis mouse models. The inverse correlation between NK activity and metastasis incidence supports a critical role for NK cells in human metastatic surveillance. However, autologous NK cell therapy has shown limited benefit in treating patients with metastatic solid tumors. Using a spontaneous metastasis mouse model of MHC-I+ breast cancer, we found that transfer of IL-15/IL-12-conditioned syngeneic NK cells after primary tumor resection promoted long-term survival of mice with low metastatic burden and induced a tumor-specific protective T cell response that is essential for the therapeutic effect. Furthermore, NK cell transfer augments activation of conventional dendritic cells (cDCs), Foxp3-CD4+ T cells and stem cell-like CD8+ T cells in metastatic lungs, to which IFN-{gamma} of the transferred NK cells contributes significantly. These results imply direct interactions between transferred NK cells and endogenous cDCs to enhance T cell activation. We conducted an investigator-initiated clinical trial of autologous NK cell therapy in six patients with advanced cancer and observed that the NK cell therapy was safe and showed signs of effectiveness. These findings indicate that autologous NK cell therapy is effective in treating established low burden metastases of MHC-I+ tumor cells by activating the cDC-T cell axis at metastatic sites.

immunology↗

bioRxiv2024

Massively Parallel Polyribosome Profiling Reveals Translation Defects of Human Disease-Relevant UTR Mutations

The untranslated regions (UTRs) of mRNAs harbor regulatory elements influencing translation efficiency. Although 3.7% of disease-relevant human mutations occur in UTRs, their exact role in pathogenesis remains unclear. Through metagene analysis, we mapped pathogenic UTR mutations to regions near coding sequences, with a focus on the upstream open reading frame (uORF) initiation site. Subsequently, we utilized massively parallel poly(ribo)some profiling to compare the ribosome associations of 6,555 pairs of wildtype and mutant UTR fragments. We identified 46 UTR variants that altered polysome profiles, with enrichment in pathogenic mutations. Both univariate analysis and the elastic net regression model highlighted the significance of motifs of short repeated sequences, including SRSF2 binding sites, as mutation hotspots that lead to aberrant translation. Furthermore, these polysome-shifting mutations exhibited considerable impact on RNA secondary structures, particularly for upstream AUG-containing 5 UTRs. Integrating these features, our model achieved high accuracy (AUROC > 0.8) in predicting polysome-shifting mutations in the test dataset. Additionally, several lines of evidence indicate that changes in uORF usage underlie the translation deficiency arising from these mutations. Illustrating this, we demonstrate that a pathogenic mutation in the IRF6 5 UTR suppresses translation of the primary open reading frame by creating a uORF. Remarkably, site- directed ADAR editing of the mutant mRNA rescued this translation deficiency. Overall, our study provides insights into the molecular mechanisms of UTR mutations and their links to clinical impacts through translation defects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/589132v2_ufig1.gif" ALT="Figure 1000"> View larger version (50K): org.highwire.dtl.DTLVardef@57217corg.highwire.dtl.DTLVardef@575410org.highwire.dtl.DTLVardef@15013bforg.highwire.dtl.DTLVardef@408840_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Multiplexed Assays of Human Disease-relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability

UTRs contain crucial regulatory elements for RNA stability, translation and localization, so their integrity is indispensable for gene expression. Approximately 3.7% of genetic variants associated with diseases occur in UTRs, yet a comprehensive understanding of UTR variant functions remains limited due to inefficient experimental and computational assessment methods. To systematically evaluate the effects of UTR variants on RNA stability, we established a massively parallel reporter assay on 6,555 UTR variants reported in human disease databases. We examined the RNA degradation patterns mediated by the UTR library in two cell lines, and then applied LASSO regression to model the influential regulators of RNA stability. We found that UA dinucleotides and UA-rich motifs are the most prominent destabilizing element. Gain of UA dinucleotide outlined mutant UTRs with reduced stability. Studies on endogenous transcripts indicate that high UA-dinucleotide ratios in UTRs promote RNA degradation. Conversely, elevated GC content and protein binding on UA dinucleotides protect high-UA RNA from degradation. Further analysis reveals polarized roles of UA- dinucleotide-binding proteins in RNA protection and degradation. Furthermore, the UA- dinucleotide ratio of both UTRs is a common characteristic of genes in innate immune response pathways, implying a coordinated stability regulation through UTRs at the transcriptomic level. We also demonstrate that stability-altering UTRs are associated with changes in biobank-based health indices, underscoring the importance of precise UTR regulation for wellness. Our study highlights the importance of RNA stability regulation through UTR primary sequences, paving the way for further exploration of their implications in gene networks and precision medicine.

genetics↗

bioRxiv2024

An unscheduled switch to endocycles induces a reversible senescent arrest thatimpairs growth of the Drosophila wing disc

A programmed developmental switch to G / S endocycles results in tissue growth through an increase in cell size. Unscheduled, induced endocycling cells (iECs) promote wound healing but also contribute to cancer. Much remains unknown, however, about how these iECs affect tissue growth. Using the D. melanogaster wing disc as model, we find that populations of iECs initially increase in size but then subsequently undergo a heterogenous arrest that causes severe tissue undergrowth. iECs acquired DNA damage and activated a Jun N-terminal kinase (JNK) pathway, but, unlike other stressed cells, were apoptosis-resistant and not eliminated from the epithelium. Instead, iECs entered a JNK-dependent and reversible senescent-like arrest. Senescent iECs promoted division of diploid neighbors, but this compensatory proliferation did not rescue tissue growth. Our study has uncovered unique attributes of iECs and their effects on tissue growth that have important implications for understanding their roles in wound healing and cancer.

developmental biology↗