Biology subjects

Chen, S. X.

Publications and source records attributed to Chen, S. X..

8 recordsLinked to original sources

bioRxiv2026

Tobacco Carcinogen Induces Persistent DNA Damage and Homologous Recombination Dysregulation in Pancreatic Ductal Carcinogenesis

Cigarette smoking is a well-established risk factor for the development of pancreatic ductal adenocarcinoma (PDAC), yet the mechanisms by which tobacco carcinogens, such as Nicotine-Derived Nitrosamine Ketone (NNK), drive pancreatic carcinogenesis remain incompletely understood. We investigated the impact of NNK on pancreatic epithelium by treating human pancreatic ductal epithelial cells (HPDE6-E6E7; H6c7) with NNK for 5 or 50 days followed by genome-wide RNA sequencing, qRT-PCR, immunofluorescence, comet assay, and {gamma}H2AX ELISA. We assessed {gamma}H2AX by immunohistochemistry in human PDAC tissue microarrays and in TCGA-PAAD datasets to identify clinical correlates. Acute NNK exposure (5 days) induced DNA double-strand breaks and activated ATM-{gamma}H2AX signaling, accompanied by increased RAD51 and BRCA2 recruitment, consistent with activation of homologous recombination (HR)-mediated DNA repair. In contrast, chronic NNK exposure (50 days) resulted in persistent DNA damage signaling and transcriptional upregulation of HR-associated genes, including RAD51, BRCA1, BRCA2, FANCD2, and PLK1. Despite sustained accumulation of RAD51- and BRCA2-positive repair foci, chronic NNK exposure was associated with altered RAD51 localization, persistent DNA damage, and chromosomal instability, suggesting dysregulation of HR repair fidelity during prolonged genotoxic stress. {gamma}H2AX expression was significantly elevated in NNK-treated cells, KRAS-driven premalignant pancreatic lesions, and pancreatic tissues from smokers. Increased {gamma}H2AX expression correlated with tumor progression and poor overall survival, while elevated RAD51 expression was associated with adverse outcomes in patients with PDAC. Collectively, these findings reveal a biphasic DNA damage response to NNK characterized by early activation of ATM-{gamma}H2AX-dependent DNA repair followed by persistent DNA damage signaling, dysregulated HR repair, and genomic instability during chronic exposure. Tobacco-induced disruption of DNA repair homeostasis may represent a key mechanism driving pancreatic carcinogenesis and supports {gamma}H2AX as a clinically relevant prognostic biomarker in tobacco-associated PDAC.

cancer biology↗

bioRxiv2026

Cost-Effective Purification of Endotoxin-Free LIF, IL-2, and IL-33

We describe a cost-effective Escherichia coli (E. coli)-based platform for producing endotoxin-free cytokines. As a proof of concept, we applied this protocol to purify four representative human and mouse cytokines - mouse leukemia inhibitory factor (mLIF), human interleukin-2 (hIL-2), and human and mouse interleukin-33 (hIL-33 and mIL-33) - and demonstrated their bioactivity to be equivalent to commercial counterparts for direct use in stem cell culture and immune cell activation, both in vitro and in vivo. Reagent costs for producing these proteins are approximately 5%-10% of commercial list prices. This platform is readily adaptable to other costly cytokines and growth factors, providing a scalable and affordable approach to accelerate research in cell biology, tissue engineering, and biomanufacturing.

bioengineering↗

bioRxiv2025

An Integrative Multi-Omics Random Forest Framework for Robust Biomarker Discovery

High-throughput technologies now produce a wide array of omics data, from genomic and transcriptomic profiles to epigenomic and proteomic measurements. Integrating these diverse data types can yield deeper insights into the biological mechanisms driving complex traits and diseases. Yet, extracting key shared biomarkers from multiple data layers remains a major challenge. We present a multivariate random forest (MRF)-based framework enhanced by a novel inverse minimal depth (IMD) metric for integrative variable selection. By assigning response variables to tree nodes and employing IMD to rank predictors, our approach efficiently identifies essential features across different omics types, even when confronted with high-dimensionality and noise. Through extensive simulations and analyses of multi-omics datasets from The Cancer Genome Atlas, we demonstrate that our method outperforms established integrative techniques in uncovering biologically meaningful biomarkers and pathways. Our findings show that selected biomarkers not only correlate with known regulatory and signaling networks but can also stratify patient subgroups with distinct clinical outcomes. The methods scalable, interpretable, and user-friendly implementation ensures broad applicability to a range of research questions. This MRF-based framework advances robust biomarker discovery and integrative multi-omics analyses, accelerating the translation of complex molecular data into tangible biological and clinical insights.

bioinformatics↗

bioRxiv2022

Sequencing by avidity enables high accuracy with low reagent consumption

We present avidity sequencing - a novel sequencing chemistry that separately optimizes the process of stepping along a DNA template and the process of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar, and yield negligible dissociation rates. We demonstrate the use of avidites as a key component of a sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing. We also show the advantages of this technology in sequencing through long homopolymers.

genomics↗

bioRxiv2021

Nuclear progesterone receptor regulates blood coagulation facilitating ovulation in zebrafish

Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates.

physiology↗

bioRxiv2021

LBH is a cancer stem cell- and metastasis-driving oncogene essential for WNT stem cell function in breast cancer

Cancer stem cells (CSCs) initiate tumors, resist treatment, and seed lethal metastases; yet CSC-specific treatments are lacking. Aggressive, treatment-resistant triple-negative breast cancers (TNBC) exhibit WNT pathway activation and are CSC enriched. Here, we show that Limb-Bud- and-Heart (LBH), a WNT/{beta}-catenin target required for normal mammary stem cell self-renewal, marks poor prognosis, stem-like TNBC, and is a key controller of breast cancer stemness. LBH is specifically expressed in tumor-initiating CD44+CD24-/low breast CSCs. LBH overexpression confers stem-like, metastatic traits on both TNBC and luminal origin, non-TNBC breast cancer cells by activating stem cell transcriptional programs. Importantly, silencing LBH potently suppresses tumor initiation and metastasis in vivo, and sensitizes TNBC cells to chemotherapy. LBH knockout in the MMTV-Wnt1 breast cancer mouse model, furthermore, revealed LBH is required for WNT-driven breast CSC expansion. Our findings identify LBH as an essential CSC driver downstream of WNT, and a new molecular target for anti-cancer stem cell therapy.

cancer biology↗

bioRxiv2020

Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

DNA sequence variants with low allele frequencies below 1% are difficult to detect and quantitate by sequencing, due to the intrinsic error of sequencing-by-synthesis (NGS). Unique molecular identifier barcodes can in principle help NGS detect mutations down to 0.1% variant allele frequency (VAF), but require extremely high sequencing depths of over 25,000x, rendering high sensitivity mutation detection out of reach for most research and clinical samples. Here, we present the multiplex blocker displacement amplification (mBDA) method to selectively enrich DNA variants by an average of 300-fold in highly multiplexed NGS settings. On a 80-plex human single nucleotide polymorphism panel, mBDA achieves a 0.019% VAF limit of detection for single nucleotide variants, using only 250x sequencing depth, and detects human cell line contamination down to 0.07%. Using this technology, we constructed a 16-plex melanoma NGS panel covering 145 actionable mutations across 9 genes, and applied it to 19 fresh/frozen tumor biopsy tissue samples with high tumor fractions. We found low VAF mutations (0.2% to 5%) in 37% of the samples (7/19, 95% confidence interval 19%-58%). These results suggest that tumor heterogeneity could be significantly more pervasive than previously recognized, and can contribute significantly to acquired drug resistance to targeted therapies. We also validate mBDA panels on clinical cell-free DNA samples from lung cancer patients.

bioengineering↗

bioRxiv2017

Simple And Multiplexed Enrichment Of Rare DNA Variants Via Sequence-Selective And Temperature-Robust Amplification

Rare DNA sequence variants hold important clinical and biological information, but are chal-lenging for existing methods (e.g. PCR, NGS) to profile in an inexpensive, multiplexed, simple-to-implement, and sequence-general way. Here, we present Blocker Displacement Amplification (BDA), a temperature-robust PCR method that selectively amplifies all sequence variants within a roughly 20 nt window by 1000-fold over wildtype sequences, allowing easy detection and quantitation of hundreds of potentials variants originally at [≤]0.1% allele frequency. BDA employs a rationally designed competitive hybridization reaction to achieve similar enrichment performance across anneal temperatures ranging from 56{degrees}C to 64{degrees}C. This temperature robustness facilitates multiplexed enrichment of many different variants across the genome, and furthermore enables the use of in-expensive and portable thermocycling instruments for rare DNA variant detection. To show the sequence generality of BDA, we demonstrated enrichment on 156 single-nucleotide variants (SNVs). BDA has been validated on multiple different PCR platforms, DNA polymerases, and sample types including clinical cell-free DNA samples collected from the blood plasma of lung cancer patients. BDA quantitation of mutation allele fraction is generally consistent with deep sequencing results.

genomics↗