Biology subjects

Do, C.

Publications and source records attributed to Do, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Combinatorial screening of nanoparticles for nose-to-brain RNA delivery to modulate neuroinflammation after traumatic brain injury

Traumatic brain injury (TBI)-induced neuroinflammation can evolve over weeks or months, contributing to ongoing secondary damage and worsening neurological recovery. RNA-based therapeutics hold great potential to regulate inflammatory signaling, but delivery to the brain remains challenging because of the blood brain barrier. Intranasal administration offers a direct non-invasive route for brain access but is often limited by low delivery efficiency. Here we constructed a combinatorial library of DNA-barcoded 103 lipid nanoparticles by varying lipid components with diverse headgroup chemistries and bioreactive moieties. A high throughput screening of these nanoparticles in vivo following intranasal administration led to the identification of top candidates with highest brain accumulation. Intranasal delivery of an antagomir targeting microRNA-9-5p loaded nanoparticle effectively reduced cerebral microRNA-9-5p levels, suppressed inflammatory markers, and improved neurological outcomes in TBI. These results demonstrate a systematic approach for optimizing intranasal lipid nanoparticle design and support the feasibility of RNA delivery to modulate neuroinflammation after brain injury.

bioengineering↗

bioRxiv2026

Chromatin architecture and physical constriction cooperate in phenotype switching and cancer cell dissemination

Phenotypic plasticity is a prominent cancer feature that contributes to metastatic potential and resistance to therapy across multiple cancer types. Cancer cell state transitions have been attributed to transcriptional programs, such as the AP1/TEAD-regulated gene network driving the mesenchymal-like (MES) phenotype. In addition, during dissemination, tumor cells are subjected to variable loads of physical mechanical pressure and constriction across transited tissue, which are thought to impact nuclear molecular crowding. How the interplay between mechanical pressure, global 3D nuclear architecture and transcriptional programs contributes to MES identity and metastatic adaptation remains unclear. Using cutaneous melanoma as a model for early dissemination, we integrate in vitro and in vivo epigenomic profiling with nanoscale imaging of cell lines and patient samples to investigate chromatin organization features underlying the MES phenotype. We find that in MES cells, CTCF is relocated from domain boundaries to regulatory regions of EMT-like genes, leading to reduced insulation, extended topological associated domains (TADs) and increased inter-domain contacts, and de novo formation of chromatin hubs. This conformational rewiring, along with loss of heterochromatin, supports nuclear deformability during invasion and dissemination. Conversely, physical constriction of melanocytic cells induces MES-like chromatin features--including CTCF repositioning and heterochromatin loss-- and promotes metastasis in vivo. Similarly, pharmacological inhibition of the heterochromatin mark H3K9me3 triggers MES characteristics and increases invasiveness. These results demonstrate that metastatic competency involves both epigenetic and structural nuclear reprogramming, enabling shifts in gene networks and physical adaptability. Our findings reveal mechanistic links between nuclear architecture and aggressive tumor behavior, identifying potential biomarkers and therapeutic targets to intercept metastatic progression.

cancer biology↗

bioRxiv2026

Predicting single-cell responses to novel genetic perturbations with optimal transport

The combination of pooled genetic perturbation screening with single-cell RNA-sequencing has allowed for large-scale profiling of single-cell transcriptional responses, providing valuable insights into gene function and cellular behavior. There is a need for computational methods capable of predicting single-cell responses to novel genetic perturbations, thereby allowing for the efficient exploration of the vast combinatorial space of potential multi-gene perturbations. Optimal transport (OT) presents a compelling framework to model this type of data given the lack of true control-perturbed cell pairings. However, existing neural OT models are generally constrained to the perturbations already seen in training, while the use of OT theory in methods capable of modeling novel genetic perturbations remains limited. To bridge this gap, we present an OT-based method to predict single-cell transcriptional responses to novel genetic perturbations. Leveraging a flexible attention-based aggregation approach, our method integrates gene representations from multiple sources of prior knowledge, spanning from functional descriptions of genes to gene-gene relationships, producing a biologically informed representation of the perturbation. Moreover, we utilize an OT-based loss to align predicted and observed perturbed cell populations, avoiding the need to assign random control-perturbed cell pairings. Experiments on three benchmark datasets demonstrate the highly competitive performance of our model compared to state-of-the-art methods. Further evaluations on differential expression analysis and genetic interaction modeling demonstrate the biological relevance and potential utility of our model across diverse applications.

bioinformatics↗

bioRxiv2025

A developmentally regulated long-range enhancer-promoter contact mediates human neural development

SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.

developmental biology↗

bioRxiv2025

Pathogen-Phage Geomapping to Overcome Resistance

The rise of antibiotic resistance has renewed interest in bacteriophages as therapeutic alternatives. However, co-evolution of phage and bacteria will naturally give rise to phage-resistant pathogens, complicating phage therapy efforts. A critical bottleneck in the production of phage therapeutics is the discovery of virulent phages against resistant pathogens. Conventional methods for discovery are time-consuming, biased, and laborious, limiting the potential for identifying suitable phage candidates. To overcome these limitations, we combined small-volume environmental sampling with 16S rRNA sequencing to identify reservoirs where bacterial hosts co-exist with their phage predators. This strategy, which we term geographical phage mapping (ge{Phi}mapping), pinpoints ecological "hotspots" for targeted phage hunting. We further developed a portable phage hunting device ({Phi}HD) that generates highly enriched phage concentrates directly from these reservoirs. By integrating ge{Phi}mapping with high-throughput enrichment, we constructed the R{Phi} library, a diverse collection of novel phages. We captured and isolated 36 new phages targeting extremely-resistant organisms across various ESKAPE pathogens when conventional phage hunting and experimental evolution approaches failed.

microbiology↗

bioRxiv2025

Phasing single-molecule nano-NOMe-seq reveals chromatin state heterogeneity in the context of transcription and long-range interactions

A central challenge in molecular biology is determining how 3D chromatin architecture, particularly enhancer-promoter looping and insulating CTCF-mediated interactions, influences gene transcription in individual cells, which has significant implications for healthy and diseased states. To overcome current limitations in imaging and genomic technologies, we developed a cluster-based phasing strategy using long read nano-NOMe-seq to link distinct CTCF binding states-- captured at the single molecule level--to the transcriptional status of genes. By stitching partially overlapping long reads and clustering them by shared GpC-accessibility patterns, we stratify CTCF into graded binding states on individual molecules, classify RNA polymerase states at promoters/gene bodies, and infer when spatially separated loci are coordinately activated and occupy loop-competent configurations on the same molecules. When applied to Sox2, Hoxa, and Klf1 regions, cluster-based nano-NOMe-seq phasing reveals how specific topologies bias polymerase behavior and multi-locus activity in ways that bulk assays or locus-engineered imaging cannot fully capture.

molecular biology↗

bioRxiv2025

Persistent chromatin alterations and gene expression reprogramming follow widespread DNA damage in glioblastoma

DNA damage from routine cellular processes or exogenous insults can have a lasting impact on gene regulation beyond genetic mutations. The prevailing paradigm for the consequences of DNA damage repair revolves around restoration of the original genetic sequence, but long-term changes in chromatin configuration, gene expression and DNA modifications have not been analyzed. We introduced numerous, simultaneous Cas9-mediated DNA double strand breaks (DSBs) at defined locations in human glioblastoma cells and tracked both non-genetic and genetic alterations over time. Megabase-scale genomic alterations that endured two weeks after the initial damage were detected, involving a shift from transiently increased intra-TAD interactions to persistent long range cis and trans contacts, alterations in gene-expression and associated large structural variations. These findings reveal that widespread DNA damage, such as chemotherapy or radiotherapy, can trigger long-term genetic and non-genetic modifications which alter cellular function and may impact tumor outcome and the emergence of resistant cells.

genomics↗

bioRxiv2025

mtgRNA-db: An annotated database of multi-target CRISPR-Cas9 guide-RNAs in the human genome

CRISPR-Cas9 guide-RNA design tools can be used to identify guide-RNAs that target single human genomic loci. However, these approaches limit their effect to a single locus. Here, we generate a database of potential individual guide-RNAs that target multiple sites in the genome at once. All guide-RNAs in this database are curated with on- and off-target quantification and enrichment scores for genomic elements such as gene elements, regulatory elements, repetitive elements, and transcription factor motifs. This tool enables rationale mass targeting of genomic loci with a single guide for functional studies. We created a web-app for user-friendly guide-RNA selection (https://modreklab.shinyapps.io/guiderna/).

bioinformatics↗

bioRxiv2025

Incorporating Hierarchical Information into Multiple Instance Learning for Patient Phenotype Prediction with scRNA-seq Data

Multiple Instance Learning (MIL) provides a structured approach to patient phenotype prediction with single-cell RNA-sequencing (scRNA-seq) data. However, existing MIL methods tend to overlook the hierarchical structure inherent in scRNA-seq data, especially the biological groupings of cells, or cell types. This limitation may lead to suboptimal performance and poor interpretability at higher levels of cellular division. To address this gap, we present a novel approach to incorporate hierarchical information into the attentionbased MIL framework. Specifically, our model applies the attention-based aggregation mechanism over both cells and cell types, thus enforcing a hierarchical structure on the flow of information throughout the model. Across extensive experiments, our proposed approach consistently outperforms existing models and demonstrates robustness in data-constrained scenarios. Moreover, ablation test results show that simply applying the attention mechanism on cell types instead of cells leads to improved performance, underscoring the benefits of incorporating the hierarchical groupings. By identifying the critical cell types that are most relevant for prediction, we show that our model is capable of capturing biologically meaningful associations, thus facilitating biological discoveries.

bioinformatics↗

bioRxiv2024

A targetable developmental program co-regulates angiogenesis and immune evasion

Ultraviolet (UV)-induced DNA mutations produce genetic drivers of cutaneous melanoma initiation and numerous neoantigens that can trigger anti-tumor immune responses in the host. Consequently, melanoma cells must rapidly evolve to evade immune detection by simultaneously modulating cell-autonomous epigenetic mechanisms and tumor-microenvironment interactions. Angiogenesis has been implicated in this process; although an increase of vasculature initiates the immune response in normal tissue, solid tumors manage to somehow enhance blood flow while preventing immune cell infiltration. By comparing the expression of transcription factors (TFs) across early-stage melanoma, naevi, and other cancer types, we found the homeodomain-containing TF HOXD13 drives a melanoblast-like developmental program, which is upregulated in melanoma and strongly correlated with angiogenesis and immune cell exclusion. Using transcriptomics, 3D chromatin profiling, and in vivo models, we demonstrate that HOXD13 upregulation promotes tumor growth in vivo by concomitantly enhancing angiogenesis and suppressing T-cell infiltration. HOXD13 orchestrates 3D chromatin contacts between distal enhancers and promoters, simultaneously activating VEGFA, SEMA3A, and CD73. VEGFA and SEMA3A remodel the tumor vasculature and CD73 elevates extracellular levels of adenosine, a vasodilator and immune suppressor that binds adenosine receptors (AdR) on endothelial and T cells. In line with these findings, HOXD13-induced growth advantage in vivo was significantly reversed by the concomitant administration of VEGFR and AdR inhibitors. By revealing a dual pro-angiogenic and immunosuppressive HOXD13-CD73/VEGF gene regulatory axis, we identify a subset of patients who might benefit from combinations of AdR and VEGFR inhibitors which are both currently being tested in clinical trials.

cancer biology↗

bioRxiv2024

A genome wide code to define cell-type specific CTCF binding and chromatin organization

CTCF-mediated chromatin folding plays a key role in gene regulation, however the mechanisms controlling chromatin organization across cell states are not fully elucidated. Comprehensive analyses reveal that CTCF binding stability and cohesin overlap in mice and humans, are regulated by species specific differences in CTCF binding site (CBS) accessibility and enrichment of motifs corresponding to expressed TFs. By analyzing TFs, we confirm the co-operativity and competitiveness of TF/CTCF binding, which we further validate by allele specific analysis of SNPs. TF motif enrichment at CTCF bound sites is determined by cell state-specific transcriptional programs, which either stabilize or destabilize CTCF binding, as reflected by changing TF concentration. To examine CTCF binding in the context of nucleosome positioning, we performed single molecule nano-NOMe-seq. Subsetting reveals a continuum of binding states for CTCF, which are differentially represented at accessible versus inaccessible CBSs. As expected, CTCF degradation leads to a progressive loss of binding and nucleosome repositioning giving profiles similar to CTCF free CBSs. We also observe a similar time dependent effect when the cohesin subcomponent, SSC1 is degraded although CTCF remains bound, indicating that cohesin mediates CTCF-associated nucleosome repositioning. Stratified analysis of CTCF signal strength and accessibility reveals that in the presence of cohesin, CTCF strength contributes to nucleosome repositioning and chromatin insulation independent of accessibility. However, cobound TFs can uncouple the relationship between signal strength and nucleosome repositioning, without affecting the connection between repositioning and insulation. These studies identify mechanisms underlying cell state-specific CTCF profiles, linked to local and long-range chromatin organization.

genomics↗

bioRxiv2024

Lysyl Oxidases are Necessary for Myometrial Contractility and On-Time Parturition in Mice

The extracellular matrix (ECM) plays a pivotal role in the maintenance of tissue mechanical homeostasis. Collagens and elastic fibers are the most predominant fibrous ECM proteins providing tissue mechanical function through covalent cross-linking which is mediated by the lysyl oxidase family of enzymes. In this study, the function of lysyl oxidases in maintaining the integrity of the extracellular matrix in the myometrium and its impact on parturition-timing was investigated. Gene and protein expression analyses demonstrate that a sub-set of the lysyl oxidase family of enzymes are highly induced in pregnant myometrium. Inhibition of the activity of the lysyl oxidase family of enzymes through {beta}-aminopropionitrile (BAPN) delays parturition in mice, in part, due to myometrial dysfunction. In BAPN treated mice, the expression of genes encoding contraction associated proteins such as connexin 43, oxytocin receptor and prostaglandin synthase 2 is significantly reduced in the myometrium compared to the untreated control mice. Proteomic analysis revealed that the composition of the ECM is altered in response to BAPN treatment which demonstrates that the inhibition of the activity of lysyl oxidases disrupted the integrity of the myometrial ECM. Our findings demonstrate that the lysyl oxidases-mediated ECM function is necessary for the myometrium to transition from a quiescent to a contractile phenotype at term for on-time parturition.

physiology↗

bioRxiv2024

Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial- mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome- wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

developmental biology↗

bioRxiv2024

Improving allele-specific epigenomic signal coverage by 10-fold using Hidden Markov Modeling and Machine Learning

Allele-specific epigenomic signals refer to differences in epigenomic patterns between the two copies, or "alleles," of a DNA region inherited from each parent. Epigenomic patterns are defined as alterations of the DNA sequence (e.g., chemical) without modifying the underlying DNA sequence (which would be referred to as "mutations"). Mapping allele-specific epigenomic signals across a genome is crucial, as some can influence gene expression, disease susceptibility, and developmental processes. However, identifying allele-specific epigenomic patterns across an entire genome is limited by the average read length (50-150 nucleotides) of short-read sequencing technologies, which are the most widely-used and affordable whole genome sequencing methods, and by the 99.9% similarity in the DNA sequences inherited from each parent. These limitations restrict the assessment of allele-specific signals to approximately 10% of the genome, potentially overlooking critical regulatory regions. In this paper, we present a highly effective machine-learning approach based on variational hidden Markov modeling, which enables the detection of allele-specific epigenomic signals across the entire genome, resulting in a 10-fold improvement in genomic coverage compared to state-of-the-art methods. We demonstrate our method on DNA methylation, a critical epigenomic regulatory signal.

genomics↗

bioRxiv2024

Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer

Here we used a series of CTCF mutations to explore CTCFs relationship with chromatin and its contribution to gene regulation. CTCFs impact depends on the genomic context of bound sites and the unique binding properties of WT and mutant CTCF proteins. Specifically, CTCFs signal strength is linked to changes in accessibility, and the ability to block cohesin is linked to its binding stability. Multivariate modelling reveals that both CTCF and accessibility contribute independently to cohesin binding and insulation, however CTCF signal strength has a stronger effect. CTCF and chromatin have a bidirectional relationship such that at CTCF sites, accessibility is reduced in a cohesin-dependent, mutant specific fashion. In addition, each mutant alters TF binding and accessibility in an indirect manner, changes which impart the most influence on rewiring transcriptional networks and the cells ability to differentiate. Collectively, the mutant perturbations provide a rich resource for determining CTCFs site-specific effects.

molecular biology↗

bioRxiv2023

The ventral hippocampus and nucleus accumbens as neural substrates for cocaine contextual memory reconsolidation

Drug craving triggered by cues that were once associated with drug intoxication is a major contributor to continued drug-seeking behaviors. Addictive drugs engage molecular pathways of associative learning and memory. Reactivated memories are vulnerable to disruption by interference with the process of reconsolidation, hence targeting reconsolidation could be a strategy to reduce cue-induced drug craving and relapse. Here we examined the circuitry of cocaine contextual memory reconsolidation and explored neuroplasticity following memory reactivation. Mice underwent chemogenetic inhibition of either nucleus accumbens (NA) neurons or the glutamatergic projection neurons from the ventral hippocampus (vHPC) to NA using inhibitory designer receptors exclusively activated by designer drugs (iDREADD). Mice underwent cocaine conditioned place preference followed by reactivation of the cocaine contextual memory. Clozapine-N-oxide (CNO) was administered after memory reactivation to inhibit either NA neurons or the accumbens-projecting vHPC neurons during the reconsolidation period. When retested 3 days later, a significant reduction in the previously established preference for the cocaine context was found in both conditions. FosTRAP2-Ai14 mice were used to identify neurons activated by cocaine memory recall and to evaluate plasticity in NA medium spiny neurons (MSNs) and vHPC pyramidal neurons upon recall of cocaine memories. Results indicate a significant increase in dendritic spine density in NA MSNs activated by cocaine memory recall, particularly of the thin spine type. Sholl analysis indicated longer dendritic length and more branching of NA MSNs after cocaine memory recall than without memory reactivation. vHPC neurons showed increased spine density, with the most robust change in stubby spines. These results implicate a circuit involving glutamatergic projections from the vHPC onto NA neurons which is necessary for the reconsolidation of cocaine memories. Interruption of cocaine memory reconsolidation reduced drug-seeking behavior.

neuroscience↗

bioRxiv2023

R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, R- loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.

genomics↗

bioRxiv2023

MethNet: a robust approach to identify regulatory hubs and their distal targets in cancer

Aberrations in the capacity of DNA/chromatin modifiers and transcription factors to bind non-coding regions can lead to changes in gene regulation and impact disease phenotypes. However, identifying distal regulatory elements and connecting them with their target genes remains challenging. Here, we present MethNet, a pipeline that integrates large-scale DNA methylation and gene expression data across multiple cancers, to uncover novel cis regulatory elements (CREs) in a 1Mb region around every promoter in the genome. MethNet identifies clusters of highly ranked CREs, referred to as hubs, which contribute to the regulation of multiple genes and significantly affect patient survival. Promoter-capture Hi-C confirmed that highly ranked associations involve physical interactions between CREs and their gene targets, and CRISPRi based scRNA Perturb-seq validated the functional impact of CREs. Thus, MethNet-identified CREs represent a valuable resource for unraveling complex mechanisms underlying gene expression, and for prioritizing the verification of predicted non-coding disease hotspots.

cancer biology↗