Biology subjects

Wu, T.-C.

Publications and source records attributed to Wu, T.-C..

12 recordsLinked to original sources

bioRxiv2025

Intron retention regulates STAT2 function and predicts immunotherapy response in lung cancer

Immunotherapy benefits only a subset of lung cancer patients, and the molecular determinants of variable outcomes remain unclear. Using long-read RNA-sequencing we mapped the landscape of alternative RNA splicing in human primary lung adenocarcinomas. We identified over 180,000 full-length mRNA isoforms, more than half of which were novel and many of which occurred in immune-related genes, particularly within the type I interferon response pathway. We discovered retained introns in the STAT2 gene that produce altered protein isoforms that regulate immune signaling and interferon responses. Furthermore, STAT2 intron retention levels predicted patient responses to checkpoint inhibitors. These findings position alternative splicing as a key regulator of tumor immune responses and reveal mRNA splicing alterations as potential biomarkers to identify patients who might respond to cancer immunotherapy.

immunology↗

bioRxiv2025

Host-specific bacterial modulation of airway gene expression and alternative splicing

The human microbiome varies extensively between individuals. While there are numerous studies investigating the effects of inter-individual differences on microbiome composition, there are few studies investigating inter-individual effects on microbial modulation of the host, or host-specific effects. To address this knowledge gap, we colonized human bronchial epithelial air-liquid interface tissue cultures generated from six different adults with one of three phylogenetically diverse bacteria and compared how each microbe differentially modulated host gene expression in each of the six donors. Microbial treatment had the strongest effect on transcription, followed by donor-specific effects. Gene pathways differed markedly in their donor- and microbe-specificity; interferon expression was highly donor-dependent while transcription of epithelial barrier and antibacterial innate immunity genes were predominantly microbially driven. Moreover, we evaluated whether microbial regulation of alternative splicing was modulated by donor. Strikingly, we found significant non-redundant, donor-specific regulation of alternative splicing exclusively in the Gram-positive commensal microbes. These findings highlight that microbial effects on the human airway epithelium are not only species-specific but also deeply individualized, scoring the importance of host context in shaping microbe-induced transcriptional and splicing responses.

microbiology↗

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗

bioRxiv2023

Protocol for primary human lung organoid-derived air-liquid interface in vitro model to study response to SARS-CoV-2

This article presents a comprehensive protocol for establishing primary human lung organoid-derived air-liquid interface (ALI) cultures from cryopreserved human lung tissue. These cultures serve as a physiologically relevant model to study human airway epithelium in vitro. The protocol encompasses lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. It also demonstrates SARS-CoV-2 infection in these cultures as an example of their utility. Quality control steps, ALI characterization, and technical readouts for monitoring virus response are included in the study. For additional details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al (https://doi.org/10.1016/j.isci.2023.107374). HighlightsO_LIHuman lung tissue dissection, embedding in OCT blocks, and tissue cryopreservation. C_LIO_LIThawing & lung tissue dissociation for lung epithelium organoid generation. C_LIO_LIOrganoid-derived air-liquid-interface cultures for the study of viral infection. C_LIO_LIBulk RNA-Seq, flow cytometry, viral titer, and imaging to follow response to virus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/557067v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@b47fbdorg.highwire.dtl.DTLVardef@2e6e60org.highwire.dtl.DTLVardef@508ac6org.highwire.dtl.DTLVardef@1c701a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

immunology↗

bioRxiv2023

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice

BackgroundThe gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mices gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. ResultsWe gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. ConclusionsThis study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

microbiology↗

bioRxiv2022

Nuclear receptor interaction protein (NRIP) as a novel actin-binding protein involved in invadosome formation for myoblast fusion

To investigate the role of nuclear receptor interaction protein (NRIP) in myoblast fusion, both the primary myoblasts from muscle-specific NRIP-knockout mice and NRIP-null C2C12 cells (KO19 cells) exhibited a significant deficit in the fusion index during myogenesis; on the other hand, overexpressed NRIP in KO19 cells could rescue myotube formation. Furthermore, NRIP was found to interact with actin directly and reciprocally that is an invadosome component for myoblast fusion. Endogenous NRIP colocalized with components of invadosome such as F-actin, Tks5, and cortactin at the tips of cells during C2C12 differentiation, and exogenous NRIP was enriched with actin at the tip of attacking cells during myogenic fusion, implying that NRIP is a novel invadosome component. Using time-lapse microscopy and cell-cell fusion assays further confirmed NRIP directly participates in cell fusion through actin. Moreover, to map the domain of NRIP-actin binding, NRIP interacted with actin either through WD40 domains directly for binding or indirectly through the IQ domain for -actinin 2 binding with actin. NRIP with actin binding was strongly correlated with invadosome formation and myotube fusion. Collectively, NRIP acts as a novel actin-binding protein through its WD40 or the IQ to form invadosomes that trigger myoblast fusion.

cell biology↗

bioRxiv2021

Quantitative imaging in whole-mount zebrafish embryos traces morphogen gradient maintenance and noise propagation in BMP signaling

Dorsoventral (DV) embryonic patterning relies on precisely controlled interpretation of morphogen signaling. In all vertebrates, DV axis specification is informed by gradients of Bone Morphogenetic Proteins (BMPs). We developed a 3D single-molecule mRNA quantification method in whole-mount zebrafish to quantify the inputs and outputs in this pathway. In combination with 3D computational modeling of zebrafish embryo development, data from this method revealed that Sizzled (Szl), shaped by BMP and Nodal signaling, kept a consistent inhibition level with Chordin (Chd) to maintain the BMP morphogen gradient. Intriguingly, BMP morphogen intrinsic expression is highly noisy at the ventral marginal layer in early zebrafish gastrula, where the gradient for DV patterning is established, which implies an unexpected role for noise in gradient shaping.

systems biology↗

bioRxiv2020

Long-read isoform sequencing reveals survival-associated splicing in breast cancer

Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is not known. We developed a long-read RNA sequencing and analytical platform that identifies and annotates full-length isoforms, and infers tumor-specific splicing events. Application of this platform to breast cancer samples vastly expands the known isoform landscape of breast cancer, identifying thousands of previously unannotated isoforms of which ~30% impact protein coding exons and are predicted to alter protein localization and function, including of the breast cancer-associated genes ESR1 and ERBB2. We performed extensive cross-validation with -omics data sets to support transcription and translation of novel isoforms. We identified 3,059 breast tumor-specific splicing events, including 35 that are significantly associated with patient survival. Together, our results demonstrate the complexity, cancer subtype-specificity, and clinical relevance of novel isoforms in breast cancer that are only annotatable by LR-seq, and provide a rich resource of immuno-oncology therapeutic targets.

genomics↗

bioRxiv2020

WaveletSEG: Automatic wavelet-based 3D nuclei segmentation and analysis for multicellular embryo quantification

Identification of individual cells in tissues, organs, and in various developing systems is a well-studied problem because it is an essential part of objectively analyzing quantitative images in numerous biological contexts. We developed a size-dependent wavelet-based segmentation method that provides robust segmentation without any preprocessing, filtering or fine-tuning steps, and is robust to the signal-to-noise ratio (SNR). The wavelet-based method achieves robust segmentation results with respect to True Positive rate, Precision, and segmentation accuracy compared with other commonly used methods. We applied the segmentation program to zebrafish embryonic development IN TOTO for nuclei segmentation, image registration, and nuclei shape analysis. These new approaches to segmentation provide a means to carry out quantitative patterning analysis with single-cell precision throughout three dimensional tissues and embryos and they have a high tolerance for non-uniform and noisy image data sets.

systems biology↗

bioRxiv2020

Physical and mental health characteristics of adults with subjective cognitive decline: A study of 3,407 people aged 18-81 years from an MTurk-based U.S. national sample

Subjective cognitive decline (SCD), or internal feelings of reduced mental capacity, is of increasing interest in the scientific, clinical, and lay community. Much of the extant literature is focused on SCD as a risk factor for Alzheimers disease in older adults, while less attention has been paid to non-cognitive health correlates of SCD across adulthood. Consequently, we investigated physical and mental health correlates of SCD in younger, middle-aged, and older adults. We recruited 3,407 U.S. residents through Amazons Mechanical Turk, an online labor market. Participants completed a 90-item self-report survey questionnaire assessing sociodemographic characteristics, physical health, sleep, depression, anxiety, loneliness, wisdom, self-efficacy, and happiness. Overall, 493/1930 (25.5%) of younger adults (18-49) and 278/1032 (26.9%) of older adults (50 or older) endorsed the SCD item. Multivariate analysis of variance and follow-up t-tests revealed worse physical and mental health characteristics in people endorsing SCD compared to those who did not, with effect sizes primarily in the medium to large range. Additionally, age did not moderate relationships between SCD and physical and mental health. Results suggest that SCD is associated with a diverse set of negative health characteristics such as poor sleep and high body mass index, and lower levels of positive factors including happiness and wisdom. Effect sizes of psychological correlates of SCD were as large as (or larger than) those of physical correlates, indicating that mental health and affective symptoms may be critical to consider when evaluating SCD. Overall, findings from this large, national U.S. sample suggest the presence of relationships between SCD and multiple psychological and perceived health factors; our results also show that SCD may be highly prevalent in both younger and older adults, suggesting that it be assessed across the adult lifespan.

neuroscience↗

bioRxiv2019

Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

Long-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

genomics↗