Biology subjects

Hu, T.

Publications and source records attributed to Hu, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

BACH2 dosage establishes the hierarchy of stemness and finetunes antitumor immunity in CAR T cells

Self-renewing stem-like T cells promote the efficacy of cancer immunotherapy and are a heterogeneous population with sub-lineages demonstrating different degrees of stemness. At the apex of this hierarchy is long-term (LT) stem-like T cells with the highest capacity of persistence, repopulation and response to immune checkpoint inhibitors (ICI). However, the pathway that establishes the hierarchy of stemness in chimeric antigen receptor (CAR) T cells and its role in antitumor efficacy of CAR T cells are unclear. Here, we demonstrate that LT stem-like differentiation and antitumor immunity of CAR T cells are dose-dependently regulated by BACH2. Pre-infusion LT stem-like CAR T cells showed epigenetic activation of BACH2 and superior antitumor response in mice and humans. After clearing tumor in vivo, LT stem-like T cells emerged as a CAR subset that transcriptionally and epigenetically upregulated BACH2 and downregulated TOX. Loss-of-function experiments revealed an essential role of BACH2 in the antitumor response of CAR T cells and the transcriptional program of LT stem-like CAR T cells. In exhaustion-prone GD2 CAR T cells with high tonic signaling, we showed that quantitative control of BACH2 protein level by a small molecule drug finetuned the degree of stemness and exhaustion in CAR T cells. Chemically inducing temporal BACH2 activation during manufacture of GD2 CAR T cells imprinted greater antitumor immunity against solid tumor after infusion. Together, we show that BACH2 dosage establishes the hierarchy of stem-like CAR T cells and can be temporally and tunably controlled in CAR T cells to optimize differentiation and antitumor immunity.

cancer biology↗

bioRxiv2025

Identifying intervention strategies from machine learning models with COALA: a counterfactual optimization framework

MotivationMachine learning (ML) models have become increasingly complex, often functioning as black boxes that limit our understanding of the features contributing to ML predictions. Common explainable AI (XAI) methods such as SHapley Additive exPlanations (SHAP) focus on feature importance but fall short in identifying interactions among features and informing targeted, personalized interventions. Counterfactuals are hypothetical events where specific variables are altered to cause a change in outcome. These causal statements can be applied to AI models to identify actionable interventions for different subjects in a population. ResultsWe propose the framework Counterfactual Optimization for Actionable interpretabiLity in AI (COALA). COALA interprets models by identifying optimal counterfactuals for each subject, which are defined as actionable changes that lead to the most positive change in predicted outcome. When applied to a gradient boosted tree model trained on the National Health and Nutrition Examination Survey (NHANES) dataset, COALA identifies different profiles of optimal counterfactuals across subjects. Features that remain constrained were able to predict the optimal counterfactual changes for a subject at 85.4%, revealing specific features that drive what a subjects optimal counterfactual is. Availability and ImplementationCode for COALA implementation, synthetic data, models trained on synthetic data, and code to replicate results and figures are available at https://github.com/brt-solo/COALA. The NHANES 2017-2018 dataset is publicly available from the National Center for Health Statistics (NCHS). The Framingham Heart Study dataset used in this study is a publicly available, Framingham-derived dataset distributed through the Massachusetts Institute of Technology OpenCourseWare (MIT OCW) repository.

bioinformatics↗

bioRxiv2025

Assay for Rapid Quantification of Capped and Tailed Intact mRNA

The rapid development of mRNA vaccines since the COVID-19 pandemic has highlighted the potential of mRNA technology for vaccine and therapeutic applications. However, evaluating mRNA quality, including its integrity, 5' capping efficiency, and 3' poly(A) tailing, remains complex and challenging. Current analytical methods provide detailed insights, but are often limited by long analysis times, complex sample preparation, and the need for specialized expertise. The 5 CapQ assay addresses these challenges and provides a novel, rapid, and straightforward method that offers a single, unique measurement of intact mRNA with both a 5'cap and 3'poly(A) tail. This 2-hour benchtop-based assay provides quantitative or relative analysis, with high accuracy and precision without extensive sample processing. The 5 CapQ assay offers a significant advancement in mRNA analytics to alleviate bottlenecks in applications including in vitro transcription (IVT) reaction optimization, post-transcriptional capping optimization, and assessment of batch-to-batch IVT bioprocess consistency.

molecular biology↗

bioRxiv2024

Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

Neocortex expansion has a concerted relationship with folding, underlying evolution of human cognitive functions. However, molecular mechanisms underlying this significant evolutionary process remains unknown. Here, using tree shrew as an outgroup of primates, we identify a new regulator CCNB1IP1, which acquired its expression before the emergence of primates. Following the evolution of cis-regulatory elements, the CCNB1IP1 expression has steadily increased over the course of primate brain evolution, mirroring the gradual increase of neocortex. Mechanistically, we elucidated that CCNB1IP1 expression can cause an increase in neural progenitors through shortening G1 phase. Consistently, the CCNB1IP1 knock-in mouse model exhibited traits associated with enhanced learning and memory abilities. Together, our study reveals how changes in CCNB1IP1 expression may have contributed to the gradual evolution in primate brain.

evolutionary biology↗

bioRxiv2024

Path2Space: An AI Approach for Cancer Biomarker Discovery Via Histopathology Inferred Spatial Transcriptomics

Spatial transcriptomics (ST) is transforming our understanding of tumor heterogeneity by enabling high-resolution, location-specific mapping of gene expression across tumors and their microenvironment. However, the associated high cost of the assay has limited cohort size and hence large-scale biomarker discovery. Here we present Path2Space, a deep learning approach that predicts spatial gene expression directly from histopathology slides. Trained on substantial breast cancer ST data, it robustly predicts the spatial expression of over 4,300 genes in independent validations, markedly outperforming existing ST predictors. Path2Space additionally accurately infers cell-type abundances in the tumor microenvironment (TME) based on the inferred ST data. Applied to more than a thousand breast tumor histopathology slides from the TCGA, Path2Space characterizes their TME on an unprecedented scale and identifies three new spatially-grounded breast cancer subgroups with distinct survival rates. Path2Space-inferred TME landscapes enable more accurate predictions of patients response to chemotherapy and trastuzumab directly from H&E slides than those obtained by existing established sequencing-based biomarkers. Path2Space thus offers a transformative, fast and cost-effective approach to robustly delineate the TME directly from their histopathology slides, facilitating the development of spatially-grounded biomarkers to advance precision oncology.

cancer biology↗

bioRxiv2024

Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR targeting a tumor epitope, is a form of adoptive cell therapy (ACT) that has exhibited promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, making them attractive targets for TCR-T cell therapy. However, class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence an epitope encompassing such mutations can bind HLA-A2 and induce T cell responses. We report post-translational modifications (PTMs) on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRASG12V peptide, rather than the unmodified epitope, may be presented in HLA-A2 by tumor cells and impact TCR recognition. Using a novel computationally guided approach, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate/demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these new TCRs. Our findings highlight the role of PTMs in immune evasion and suggest identifying and targeting such modifications should make effective ACTs available for a substantially greater range of tumors than the current therapeutic landscape. One-sentence summaryTumor cell methylation of KRASG12V epitope in HLA-A2 permits immune evasion, and new TCRs were generated to overcome this with engineered cell therapy.

immunology↗

bioRxiv2024

Efficiently Constructing Complete Genomes with CycloneSEQ to Fill Gaps in Bacterial Draft Assemblies

BackgroundCurrent microbial sequencing relies on short-read platforms like Illumina and DNBSEQ, favored for their low cost and high accuracy. However, these methods often produce fragmented draft genomes, hindering comprehensive bacterial function analysis. CycloneSEQ, a novel long-read sequencing platform developed by BGI-Research, its sequencing performance and assembly improvements has been evaluated. FindingsUsing CycloneSEQ long-read sequencing, the type strain produced long reads with an average length of 11.6 kbp and an average quality score of 14.4. After hybrid assembly with short reads data, the assembled genome exhibited an error rate of only 0.04 mismatches and 0.08 indels per 100 kbp compared to the reference genome. This method was validated across 9 diverse species, successfully assembling complete circular genomes. Hybrid assembly significantly enhances genome completeness by using long reads to fill gaps and accurately assemble multi-copy rRNA genes, which unable be achieved by short reads solely. Through data subsampling, we found that over 500 Mbp of short-read data combined with 100 Mbp of long-read data can result in a high-quality circular assembly. Additionally, using CycloneSEQ long reads effectively improves the assembly of circular complete genomes from mixed microbial communities. ConclusionsCycloneSEQs read length is sufficient for circular bacterial genomes, but its base quality needs improvement. Integrating DNBSEQ short reads improved accuracy, resulting in complete and accurate assemblies. This efficient approach can be widely applied in microbial sequencing.

genomics↗

bioRxiv2024

Proteomics Landscape of Triple-Negative Apocrine Breast Carcinoma Reveals Molecular Mechanisms of Tumorigenesis and Characteristics of Chemo-insensitivity

Triple-negative apocrine carcinoma (TNAC) is an exceptionally rare, chemotherapy-insensitive subtype of triple-negative breast cancer (TNBC) characterized by androgen receptor (AR) positivity and low proliferative activity. Despite poor chemotherapy response, TNAC exhibits favorable long-term survival, suggesting distinct resistance mechanisms. Here, we integrate mass spectrometry-based proteomics and whole-exome sequencing (WES) to characterize TNACs molecular features. We identify progressive overexpression of PI3K/AKT and AR signaling proteins, along with ECM remodeling and upregulation of GTPase-related proteins, highlighting TNACs invasive potential. WES reveals somatic mutations in ECM-related genes (COL18A1) and immune-associated genes (C3, NRDC), implicating the tumor microenvironment. Unlike TNBC, post-chemotherapy TNAC proteomics shows mixed ribosomal regulation and heightened inflammation, rather than reliance on proliferation, suggesting a need for targeted metabolic and immune therapies. Our findings support PI3K/AKT inhibitors and AR antagonists, potentially alongside GTPase inhibitors, metabolic disruptors, and immune therapy, as alternative strategies to standard chemotherapy in TNAC.

cancer biology↗

bioRxiv2024

Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation

The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. We discovered both divergent and conserved features of tree shrew DNA methylation compared to that of other mammals. DNA methylation levels of promoter and gene body regions are negatively correlated with gene expression, consistent with patterns in other mammalian brains studied. Comparing DNA methylation patterns of the female and male X chromosomes, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire X chromosome in females. Our data suggests that the female X hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression of tree shrews. However, we identified a putative regulatory region in the 5 end of the X inactive specific transcript (Xist) gene, a key gene for XCI, whose pattern of differential DNA methylation strongly relate to its differential expression between male and female tree shrews. We show that differential methylation of this region is conserved across different species. Moreover, we provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Our study offers novel information on genomic DNA methylation of tree shrews, as well as insights into the evolution of X chromosome regulation in mammals.

genomics↗

bioRxiv2024

Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used fresh frozen tissue samples from triple-negative breast cancer (TNBC) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse phase chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

cancer biology↗

bioRxiv2024

Haploid-phased chromosomal telomere to telomere genome assembly of Uncaria rhynchophylla accelerating gene mining on the biosynthesis of medicinal alkaloids

Recorded traditionary medicinal plants contain important resources, including many natural medicinal alkaloids, for new medicine discovery and development. Uncaria genus is such a woody plant, with a high medicinal value in alkaloids, e.g. (iso)rhynchophylline. Natural alkaloids contents usually vary between germplasms and are affected by the growth environment, which requires a genomics solution to understand the genetic and environmental factors that influence alkaloid production in more detail. Here, we have dissected the haploid-resolved chromosomal T2T genome assembly of Uncaria rhynchophylla with a size of [~]634 Mb and contig N50 of 26 Mb using PacBio HiFi long-reads and Hi-C and anchored the contigs on 22 pairs of confirmed chromosomes. This genome contains 56% repeat sequences and [~]2,9000 protein-encoding genes. U. rhynchophylla diverged from a common ancestor shared with Coffea around 20 million years ago and contains expanded and contracted gene families associated with secondary metabolites and plant-associated defenses. We constructed the pathway for (iso)rhynchophylline biosynthesis with genes mined from the genome and comparative transcriptomes. 53 alkaloids out of 2,578 metabolites were identified in (iso)rhynchophylline biosynthesis, where eight differentially expressed genes were the key for regulating the catalytic steps leading to alkaloid abundance difference between tissues. The chromosome-level genome and pathways of (iso)rhynchophylline constructed in this study provide a genetic basis and guidance for further breeding improvements and the development of pharmaceutical alkaloids.

genomics↗

bioRxiv2024

Automated muscle path calibration with gradient-specified optimization based on moment arm

ObjectiveMuscle path modeling is more than just routing a cable that visually represents the muscle, but rather it defines how moment arms vary with different joint configurations. The muscle moment arm is the factor that translates muscle force into joint moment, and this property has an impact on the accuracy of musculoskeletal simulations. However, it is not easy to calibrate muscle paths based on a desired moment arm, because each path is configured by various parameters while the relations between moment arm and both the parameters and joint configuration are complicated. MethodsWe tackle this challenge in the simple fashion of optimization, but with an emphasis on the gradient; when specified in its analytical form, optimization speed and accuracy are improved. ResultsWe explain in detail how to differentiate the enormous cost function and how our optimization is configured, then we demonstrate the performance of this method by fast and accurate replication of muscle paths from a state-of-the-art shoulder-arm model. Conclusion and SignificanceAs long as the muscle is represented as a cable wrapping around obstacles, our method overcomes difficulties in path calibration, both for developing generic models and for customizing subject-specific models.

bioengineering↗

bioRxiv2024

Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-{beta} expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-{beta} induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-{beta}, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-{beta} mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-{beta} via mitochondrial dsDNA release.

immunology↗

bioRxiv2024

ZNF574 is a Quality Control Factor For Defective Ribosome Biogenesis Intermediates

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. This complexity creates a large genetic space in which pathogenic mutations can occur. "Dead-end" ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). Loss of ZNF574 results in the accumulation of faulty biogenesis intermediates that interfere with global ribosome production, further emphasizing the role of RASP in protein homeostasis and cellular health.

cell biology↗

bioRxiv2024

Multi-Niche Human Bone Marrow On-A-Chip for Studying the Interactions of Adoptive CAR-T Cell Therapies with Multiple Myeloma

Multiple myeloma (MM), a cancer of bone marrow plasma cells, is the second-most common hematological malignancy. However, despite immunotherapies like chimeric antigen receptor (CAR)-T cells, relapse is nearly universal. The bone marrow (BM) microenvironment influences how MM cells survive, proliferate, and resist treatment. Yet, it is unclear which BM niches give rise to MM pathophysiology. Here, we present a 3D microvascularized culture system, which models the endosteal and perivascular bone marrow niches, allowing us to study MM-stroma interactions in the BM niche and model responses to therapeutic CAR-T cells. We demonstrated the prolonged survival of cell line-based and patient-derived multiple myeloma cells within our in vitro system and successfully flowed in donor-matched CAR-T cells. We then measured T cell survival, differentiation, and cytotoxicity against MM cells using a variety of analysis techniques. Our MM-on-a-chip system could elucidate the role of the BM microenvironment in MM survival and therapeutic evasion and inform the rational design of next-generation therapeutics. TEASERA multiple myeloma model can study why the disease is still challenging to treat despite options that work well in other cancers.

cancer biology↗

bioRxiv2024

Non-invasive and painless mid-infrared modulation increases collagen in human and mouse skin

Stimulating collagen production in skin helps to enhance vitality while decelerating aging- associated processes in skin tissue. However, current approaches to enhancing collagen production are commonly limited by accompanying pain and trauma. Here, we report that mid-infrared modulation (MIMO) at an intensity of 70 mW/cm2 promotes collagen production in human or mouse skin in vivo without generating excessive heat. We found that protein levels of the collagen- degrading endopeptidase, MMP-1, were decreased in the skin of mice following MIMO treatment, whereas the collagen synthesis-related factors, TGF-{beta}, HSP47, and HSP70, were all increased. In addition, MIMO stimulated collagen secretion in human dermal fibroblasts in vitro. This work demonstrates that MIMO is an effective, non-invasive, and painless intervention for in vivo enhancement of collagen production in the skin. One Sentence SummaryMid-infrared modulation promotes collagen production

molecular biology↗

bioRxiv2024

Spatial Morphoproteomic Features Predict Uniqueness of Immune Microarchitectures and Responses in Lymphoid Follicles

Multiplex imaging technologies allow the characterization of single cells in their cellular environments. Understanding the organization of single cells within their microenvironment and quantifying disease-status related biomarkers is essential for multiplex datasets. Here we proposed SNOWFLAKE, a graph neural network framework pipeline for the prediction of disease-status from combined multiplex cell expression and morphology in human B-cell follicles. We applied SNOWFLAKE to a multiplex dataset related to COVID-19 infection in humans and showed better predictive power of the SNOWFLAKE pipeline compared to other machine learning and deep learning methods. Moreover, we combined morphological features inside graph edge features to utilize attribution methods for extracting disease-relevant motifs from single-cell spatial graphs. The underlying subgraphs were further analyzed and associated with disease status across the dataset. We showed that SNOWFLAKE successfully extracted significant low dimensional embedding from subgraphs with a clear separation between disease status and helped characterize unique cellular interactions in the subgraphs. SNOWFLAKE is a generalizable pipeline for the analysis of multiplex imaging data modality by extracting disease-relevant subgraphs guided by graph-level prediction.

bioinformatics↗

bioRxiv2023

Unveiling Elevated Spontaneous Mutation Rates in Phyllostachys edulis (Moso Bamboo) through Whole Genome Sequencing (WGS) and Investigating the Impact of Atmospheric and Room Temperature Plasma (ARTP) Induced Mutagenesis

Moso bamboo, recognized for its wide distribution and economic importance, encounters challenges in varietal enhancement due to its protracted sexual reproduction cycle. This study employed whole-genome resequencing to uncover spontaneous mutations in Moso bamboo and investigated mutagenesis using atmospheric and room temperature plasma (ARTP). Through the sequencing results, we identified the population of flowering bamboo as an asexual breeding line. Notably, the flowering Moso bamboo population, exclusively derived from asexual reproduction, exhibited a high spontaneous mutation rate (4.54 x 10-4 to 1.15 x 10-3/bp) during sexual reproduction, considering parental and cross-pollination effects. Genetic disparities between offspring and parents exhibited a bimodal distribution, indicating a substantial cross-pollination rate. ARTP mutagenesis increased structural variations in offspring, while changes in SNPs and INDELs were less pronounced. Sanger sequencing validated a gene subset, providing a foundation for spontaneous mutation rate investigation via whole-genome sequencing. These insights, particularly from mutagenized offspring sequencing, contribute to Moso bamboo breeding strategies. HighlightMoso bamboo breeding revolutionized--high spontaneous mutations in asexually derived flowering population. ARTP mutagenesis boosts structural variations, shaping innovative breeding approaches.

genetics↗