Biology subjects

Lee, A. H.

Publications and source records attributed to Lee, A. H..

12 recordsLinked to original sources

bioRxiv2026

Accelerated discovery of thermostable vaccines using data-efficient AI

The inherent instability of mRNA- lipid nanoparticles (LNPs) necessitates ultra-cold storage, creating significant barriers for global distribution and limiting their broader application in advanced delivery systems. Solid-state, water-free formulations offer a promising solution by enhancing thermostability and enabling integration into emerging delivery modalities such as microneedle (MN) patches. Prior efforts to stabilize mRNA-LNPs have been constrained by narrow formulation scope and low-throughput screening methods. Here, we introduce AGENT (Algorithm-Guided Experimental design for lipid Nanoparticle Thermostabilization), an AI-driven framework that couples high-throughput experimentation with Bayesian optimization to rapidly identify thermostable mRNA-LNP formulations. Manual exploration of the formulation space required months of screening and yielded suboptimal candidates. In contrast, AGENT extracted maximal information from sparse experimental datasets, enabling efficient formulation optimization in only six iterations completed within one month. Using AGENT, we stabilized mRNA vaccines with diverse LNPs, including those in clinical use, into solid state formulations that retained 100% bioactivity after storage at 37 degree C for over two months. The thermostable vaccines induced antigen-specific IgG and germinal center B cell responses that were non-inferior to those elicited by freshly prepared soluble vaccines. The solid-state formulations were further incorporated into dissolvable MN patches and administered to rodents and nonhuman primates, yielding comparable neutralizing antibody titers compared to conventional intramuscular delivery of fresh vaccines. To our knowledge, this study presents the first demonstration of AI-driven design of thermostable RNA vaccines, offering a scalable, cold-chain-free solution for global immunization. By addressing both stability and delivery challenges, AGENT provides a potentially transformative platform for developing accessible next-generation therapeutics.

bioengineering↗

bioRxiv2026

Atypical BlaIR Two-Component System in Pseudomonas aeruginosa Regulates Virulence but not β-Lactam Resistance

With the rise of antimicrobial resistance, anti-virulence therapeutics are a viable alternative to circumvent resistance pressures. Hypothetical genes and proteins are an under-studied source of potential virulence factor targets. We performed bioinformatic analyses to identify conserved hypothetical genes enriched in pathogenic Pseudomonas aeruginosa but not in non-pathogenic strains. This analysis identified an atypical BlaIR system, which we named pvmSR, that regulated P. aeruginosa virulence in a Caenorhabditis elegans infection model. This is in contrast with the typical BlaIR system from Staphylococcus aureus, which regulates resistance to {beta}-lac-tam antibiotics. The{Delta} pvmSR mutant showed reduced virulence in a C. elegans slow-killing assay. To understand how PvmSR regulated virulence in vivo, we performed dual RNA-seq to analyze transcriptomic changes in both C. elegans and P. aeruginosa. We found that C. elegans responded to P. aeruginosa {Delta}pvmSR infection by decreasing expression of lysosome and phagocytosis pathways. In P. aeruginosa {Delta}pvmSR, we observed decreased gene expression of several known virulence factors including the hydrogen cyanide synthase, hcnC, and heparinase, hepP. Additionally, we observed dysregulation in genes important for quorum sensing and biofilm formation. Collectively, our findings indicated that PvmSR contributed to virulence regulation and may serve as a potential anti-virulence target.

microbiology↗

bioRxiv2026

A class act: HDAC1-Malat1 regulates MDSC apoptosis and cell cycling to decrease suppression of T cells

Myeloid derived suppressor cells (MDSCs) are key players in the immune-suppressed tumor microenvironment (TME) and significantly contribute to immune checkpoint inhibition (ICI) resistance, making them favorable targets for cancer immunotherapy. Epigenetic reprogramming of MDSCs using histone deacetylase (HDAC) inhibitors shows promise to sensitize the TME to ICIs. However, the molecular mechanism of HDAC inhibition in MDSCs has yet to be elucidated. Murine and human MDSC models treated with Entinostat revealed that the long non-coding RNA Malat1 downregulates pSTAT3 and decreases MDSC-mediated suppression of T cell proliferation. Through HDAC inhibitor screens, we identified HDAC1 as preferentially regulating Malat1 expression, STAT3 activation, and MDSC suppression. We also show that HDAC1 inhibition increases MDSC apoptosis by shifting pro-vs. anti-apoptotic signals and increases G0/G1 cell cycle arrest via decreasing G1-S transition cyclin-CDK complexes. Collectively, our findings provide a multi-pronged mechanism of HDAC inhibition in MDSCs that inform the development of future rational combination therapies. One Sentence SummaryHDAC1 inhibition in MDSCs increases Malat1, decreases pSTAT3, induces apoptosis/cell cycle arrest, and decreases suppression of T cells

immunology↗

bioRxiv2025

Biochemical engineering of 5hmdC-DNA using a Tet3 double-mutant

5-Hydroxymethyl-2-deoxycytidine (5hmdC) is an important epigenetic marker involved in gene regulation and DNA demethylation. It has potential use as a biomarker for cancer and other diseases due to its significant depletion in various cancers and disease models. This research aimed to develop a reliable and efficient method for generating 5hmdC-containing DNA, addressing limitations in existing techniques. We created a Tet3 stalling mutant that converts 5-methyl-2'-deoxycytidine (5mdC) into a mixture of 5hmdC and 5-formyl-2'-deoxycytidine (5fdC), followed by a reduction step to convert 5fdC to 5hmdC, ensuring a pure 5hmdC state within the CpG context. This method can convert any PCR product, synthetic oligos, and entire genomes into 5hmdC-modified DNA. The principal results demonstrate high specificity and efficiency, providing a robust tool for epigenetic research, cancer diagnostics, and protein binding assays. Additionally, our technique offers 5hmdC-DNA for functional studies and as standards for diagnostic assays.

biochemistry↗

bioRxiv2025

Time-course Transcriptomics Reveals the Impact of Treponema pallidum on Microvascular Endothelial Cell Function and Phenotype

Syphilis, caused by Treponema pallidum subsp. pallidum, is an urgent global public health threat. Syphilis vaccine development has been impeded by limited understanding of the molecular mechanisms that enable T. pallidum to establish and maintain infection. The vascular endothelium is critical for T. pallidum attachment, dissemination, and host immune response initiation; however, the molecular details of T. pallidum-endothelial interactions are incompletely understood. To enhance understanding, we performed time-course transcriptomic profiling on T. pallidum-exposed brain microvascular endothelial cells. These analyses showed T. pallidum exposure altered pathways related to extracellular matrix, growth factors, integrins, and Rho GTPases. The induced transcriptional response was consistent with endothelial to mesenchymal transition, a process involved in fetal development and vascular dysfunction. This study provides a comprehensive understanding of the molecular responses of endothelial cells to T. pallidum and identified the host pathways that might cause syphilis disease symptoms, information that could aid in syphilis vaccine design. Highlights{square} Exposure of microvascular endothelial cells to Treponema pallidum subsp. Pallidum significantly alters the endothelial cell transcriptome {square}Signaling pathways related to extracellular matrix organization, growth factors, integrins, and Rho GTPases were overrepresented for genes differentially expressed in T. pallidum-exposed endothelial cells {square}Exposure to T. pallidum induces pathways and factors consistent with endothelial to mesenchymal transition, a host process central to development that may explain the devastating effects of congenital infection {square}T. pallidum exposure induces expression of Snail, the main transcription factor associated with the process of endothelial to mesenchymal transition O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/651946v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@e121beorg.highwire.dtl.DTLVardef@6e4caeorg.highwire.dtl.DTLVardef@16c3f03org.highwire.dtl.DTLVardef@19cf020_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Dynamic Adaptation in Extant Porins Facilitates Antibiotic Tolerance in Energetic Escherichia coli

Bacteria can tolerate antibiotics despite lacking the genetic components for resistance. The prevailing notion is that tolerance results from depleted cellular energy or cell dormancy. In contrast to this view, many cells in the tolerant population of Escherichia coli can exhibit motility - a phenomenon that requires cellular energy, specifically, the proton-motive force (PMF). As these motile-tolerant cells are challenging to isolate from the heterogeneous tolerant population, their survival mechanism is unknown. Here, we discovered that motile bacteria segregate themselves from the tolerant population under micro-confinement, owing to their unique ability to penetrate micron-sized channels. Single-cell measurements on the motile-tolerant population showed that the cells retained a high PMF, but they did not survive through active efflux alone. By utilizing growth assays, single-cell fluorescence studies, and chemotaxis assays, we showed that the cells survived by dynamically inhibiting the function of existing porins in the outer membrane. A drug transport model for porin-mediated intake and efflux pump-mediated expulsion suggested that energetic tolerant cells withstand antibiotics by constricting their porins. The novel porin adaptation we have uncovered is independent of gene expression changes and may involve electrostatic modifications within individual porins to prevent extracellular ligand entry.

microbiology↗

bioRxiv2023

Dynamic gene expression analysis reveals distinct severity phases of immune and cellular dysregulation in COVID-19

BackgroundCOVID-19 patients experience dynamic changes in immune and cellular function over time with potential clinical implications. However, there is insufficient research investigating, on a gene expression level, the mechanisms that become activated or suppressed over time as patients deteriorate or recover, which can inform use of repurposed and novel drugs as therapies. ObjectiveTo investigate longitudinal changes in gene expression profiles throughout the COVID-19 disease timeline. MethodsThree-hundred whole blood samples from 128 adult patients were collected during hospitalization from COVID-19, with up to five samples per patient. Transcriptome sequencing (RNA-Seq), differential gene expression analysis and pathway enrichment was performed. Drug-gene set enrichment analysis was used to identify FDA-approved medications that could inhibit critical genes and proteins at each disease phase. Prognostic gene-expression signatures were generated using machine learning to distinguish 3 disease stages. ResultsSamples were longitudinally grouped by clinical criteria and gene expression into six disease phases: Mild, Moderate, Severe, Critical, Recovery, and Discharge. Distinct mechanisms with differing trajectories during COVID-19 hospitalization were apparent. Antiviral responses peaked early in COVID-19, while heme metabolism pathways became active much later during disease. Adaptive immune dysfunction, inflammation, and metabolic derangements were most pronounced during phases with higher disease severity, while hemostatic abnormalities were elevated early and persisted throughout the disease course. Drug-gene set enrichment analysis predicted repurposed medications for potential use, including platelet inhibitors in early disease, antidiabetic medications for patients with increased disease severity, and dasatinib throughout the disease course. Disease phases could be categorized using specific gene signatures for prognosis and treatment selection. Disease phases were also highly correlated to previously developed sepsis endotypes, indicating that severity and disease timing were significant contributors to heterogeneity observed in sepsis and COVID-19. ConclusionsHigher temporal resolution of longitudinal mechanisms in COVID-19 revealed multiple immune and cellular changes that were activated at different phases of COVID-19. Understanding how a patients gene expression profile changes over time can permit more accurate risk stratification of patients and provide time-dependent personalized treatments with repurposed medications. This creates an opportunity for timely intervention before patients transition to a more severe phase, potentially accelerating patients to recovery.

immunology↗

bioRxiv2020

The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa

The bacterial stringent stress response, mediated by the signaling molecule guanosine tetra-phosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. RNA-Seq was used to investigate the transcriptome of a ppGpp-deficient strain under non-stressful nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold-change cut-off {+/-}1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity towards red blood cells, in a stringent response mutant, appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model, showed increased survival rates of the aprA mutant consistent with previous observations that stringent-response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria. Significance StatementMicroorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under non-stressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent-response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

microbiology↗

bioRxiv2019

IRIS: Big data-informed discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing

Aberrant alternative splicing (AS) is widespread in cancer, leading to an extensive but largely unexploited repertoire of potential immunotherapy targets. Here we describe IRIS, a computational platform leveraging large-scale cancer and normal transcriptomics data to discover AS-derived tumor antigens for T-cell receptor (TCR) and chimeric antigen receptor T-cell (CAR-T) therapies. Applying IRIS to RNA-Seq data from 22 glioblastomas resected from patients, we identified candidate epitopes and validated their recognition by patient T cells, demonstrating IRISs utility for expanding targeted cancer immunotherapy.

bioinformatics↗

bioRxiv2018

Comparative Multi-scale Hierarchical Structure of the Tail, Plantaris, and Achilles Tendons in the Rat

Rodent tendons are widely used to study human pathology, such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and the multi-scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achillles tendons, compare to that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definition of fascicle and fiber are often dependent on the diameter size and not their characteristic features, which impairs the ability to compare across species where the size of the fiber and fascicle might change with animal size and tendon function. Thus, the objective of the study was to select a single species that is widely used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales. This study was designed including, histology, SEM, and confocal imaging. We confirmed that rat tendons do not contain fascicles, and thus the fiber is the largest tendon subunit in the rat. In addition, we provided a structurally-based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed tendons. In all rat tendons studied, the fiber diameters were consistently 10-50 {micro}m, and this diameter appears to be conserved across larger species. Specific recommendations were made for the strengths and limitations of each rat tendon as tendon research models. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue engineered constructs.

bioengineering↗

bioRxiv2018

Yeast Genomic Screens Identify Kinesins as Potential Targets of the Pseudomonas syringae Type III Effector, HopZ1a

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as promiscuous individual T3SEs that can have multiple host targets. To overcome these challenges, we conducted heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. We screened 75 T3SEs from the plant pathogen Pseudomonas syringae and identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media, including the acetyltransferase HopZ1a. We focused our further analysis on HopZ1a, which interacts with plant tubulin and alters microtubule networks. We first performed a Pathogenic Genetic Array (PGA) screen of HopZ1a against ~4400 yeast carrying non-essential mutations and found 95 and 10 deletion mutants which reduced or enhanced HopZ1a toxicity, respectively. To uncover putative HopZ1a host targets, we interrogated both the genetic- and physical-interaction profiles of HopZ1a by identifying yeast genes with PGA profiles most similar (i.e. congruent) to that of HopZ1a, performing a functional enrichment analysis of these HopZ1a-congruent genes, and by analyzing previously described HopZ physical interaction datasets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1.\n\nARTICLE SUMMARYBacterial pathogens utilize secretion systems to directly deliver effector proteins into host cells, with the ultimate goal of promoting pathogen fitness. Despite the central role that effectors play in infection, the molecular function and host targets of most effectors remain uncharacterized. We used yeast genomics and protein interaction data to identify putative virulence targets of the effector HopZ1a from the plant pathogen Pseudomonas syringae. HopZ1a is an acetyltransferase that induces plant microtubule destruction. We showed that HopZ1a acetylated plant kinesin proteins known to regulate microtubule networks. Our study emphasizes the power of yeast functional genomic screens to characterize effector functions.

microbiology↗

bioRxiv2018

Evaluating Plastic Deformation and Damage as Potential Mechanisms for Tendon Inelasticity using a Reactive Modeling Framework

Inelastic behaviors, such as softening, a progressive decrease in modulus before failure, occur in tendon andare important aspect in degeneration and tendinopathy. These in elastic behaviors are generally attributed to two potential mechanisms: plastic deformation and damage. However, it is not clear which is primarily responsible.In this study, we evaluated these potential mechanisms of tendon in elasticity by using a recently developed reactive in elasticity model (RIE), which is a structurally-inspired continuum mechanics frame work that models tissue in elasticity based on the molecular bond kinetics. Using RIE, we formulated two material models, one specific toplastic deformation and the other to damage. The models were independently fit to published experimental tensiletests of rat tail tendons. We quantified the inelastic effects and compared the performance of the two models infitting the mechanical response during loading, relaxation, unloading, and reloading phases. Additionally, we validated the models by using the resulting fit parameters to predict an independent set of experimental stress-straincurves from ramp-to-failure tests. Overall, the models were both successful in fitting the experiments and predicting the validation data. However, the results did not strongly favor one mechanism over the other. As a result, to distinguish between plastic deformation and damage, different experimental protocols will be needed. Nevertheless, these findings suggest the potential of RIE as a comprehensive framework for studying tendon inelastic behaviors.

bioengineering↗