Biology subjects

Im, H.

Publications and source records attributed to Im, H..

13 recordsLinked to original sources

bioRxiv2026

Mechanistic and machine learning models design human gut consortia that robustly inhibit Clostridioides difficile

Identifying design principles for robust inhibition of human pathogens is a major goal of microbiome engineering. By building synthetic microbial communities from the bottom-up guided by Bayesian active learning, we investigate the Clostridioides difficile growth landscape across thousands of species-metabolite conditions. Mechanistic consumer resource and machine learning models uncover significant interactions linking metabolites and species, and exhibit concordance with microbial interactions identified in human microbiome datasets. Guided by machine learning and mechanistic models, we elucidate microbial communities capable of robustly inhibiting C. difficile across diverse nutrient environments in vitro. Metabolomic profiling identified sorbitol, mannitol, and proline as key metabolites mediating community-driven C. difficile inhibition. Model-optimized communities significantly reduced C. difficile colonization in the murine gut whereas a model-designed non-inhibitory community did not exhibit this property. Communities display consistent metabolomic patterns in vitro and in vivo, suggesting that resource competition is a design principle of robust inhibition. Together, these findings establish an integrated experimental and computational framework for the rational design of microbial communities that robustly suppress pathogens across diverse environmental contexts.

systems biology↗

bioRxiv2026

Do drugs with biliary toxicity cause cholangiocarcinoma?

AO_SCPLOWBSTRACTC_SCPLOWMany commonly used therapeutic drugs cause biliary toxicity, but it is unclear if they are directly responsible for the increasing incidence of cholangiocarcinoma (CCA). We tested experimentally and analyzed through a cohort approach whether drugs, such as the commonly used antibiotic Augmentin, which is a poster-child of biliary toxicity, are causally linked to CCA development. Using sophisticated analytical tools in cholangiocytes, including single extracellular vesicle (EV) analysis, we found no evidence that Augmentin increases the cholangiocyte malignancy marker YAP1 or phospho-YAP1. Furthermore, we analyzed the CCA incidence in our healthcare system and determined it to be 0.0932% (Augmentin group) and 0.0799% (amoxicillin control group). Although the Augmentin group showed a numerically higher CCA incidence, the association did not reach statistical significance (RR = 1.1669, 95% CI 0.6200-2.1961; Fishers exact test, P = 0.7493). Similarly, we found no evidence for cholangiocarcinoma development with other commonly used drugs, including chlorpromazine, floxuridine, 5-fluorouracil, flucloxacillin and terbinafine. We conclude that there is no direct causal relationship between clinical Augmentin doses and CCA development.

cancer biology↗

bioRxiv2025

Dynamic Shielding and Allosteric Modulation of Erythropoietin by Glycosylation

Glycosylation is a ubiquitous and essential post-translational modification that regulates protein structure, solubility, and function. Yet, the mechanisms by which glycans modulate the physicochemical properties of protein surfaces remain incompletely understood. Erythropoietin (EPO), a therapeutic glycoprotein with three N-glycosylation sites, provides a tractable model for dissecting site-specific glycan effects on protein functions. Here, we employ glycan-focused enhanced molecular dynamics simulations, specifically generalized replica exchange with solute tempering (gREST) to overcome the limited sampling of conventional MD and capture the extensive conformational heterogeneity of glycans. Our results demonstrate that glycan effects are highly non-additive: specific combinations of glycosylation sites yield emergent structural outcomes through spatial and dynamical cooperativity. Among them, the N83-linked glycan plays a dominant role in shielding a hydrophobic surface helix, thereby reducing local solvent-accessible hydropathy. Strikingly, the extent of this glycan-mediated surface masking quantitatively correlates with experimentally measured retention times in hydrophobic interaction chromatography, establishing a functional link between molecular-scale shielding and macroscopic behavior. These findings reveal that N-glycans modulate protein surfaces not only through local steric occlusion but also via long-range allosteric effects, providing a new framework for understanding and engineering glycoprotein properties.

biophysics↗

bioRxiv2025

Engineered ADARs enable single-nucleotide resolution DNA A-to-G editing without bystander effects

The adenine base editor (ABE), which enables A*T-to-G*C base conversion, has emerged as a powerful tool for therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here, we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9(H840A), with the deaminase domain of ADAR, which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a specially designed target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3-end protection of the tagRNA, further enhances the editing activity of snuABE in human cells. Moreover, snuABE exhibits significantly reduced DNA off-target activity, highlighting its potential as a safer and more precise base editing technology for therapeutic applications.

bioengineering↗

bioRxiv2025

Alterations in Nutrient Availability in the Lungs During Streptococcus pneumoniae-Induced Pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia. Importantly, the extent and impact of changes in the infected airway on bacterial nutrient availability and gene expression are not known. Utilizing untargeted UPLC-ESI-MS/MS metabolomics, we comprehensively characterized the metabolic landscape in the airway across early, mid, and severe stages of pneumococcal pneumonia. This revealed that dynamic shifts in metabolites occurred during pneumonia, with an initial influx of metabolites at the early stage, followed by declines as the disease progressed. Specific host metabolic perturbations were indicative of purine dysregulation, cellular stress, and outright tissue injury. Levels of glucose, a known modulator of pneumococcal capsule production, were highest at early disease stage, and then declined as the disease progressed, overlaying general metabolite trends. Concurrent bacterial transcriptome profiling was performed using a NanoString nCounter custom panel of 66 genes selected for their importance to metabolism, virulence, and stress response; 9% of which had disease-stage significant differences in gene expression. This analysis revealed remarkably high expression of spxB, the gene encoding pyruvate oxidase, at the severe stage of pneumonia compared to the mid-stage pneumonia, consistent with a drop in glucose levels and indicative of a shift towards mixed fermentation and the increased production of hydrogen peroxide. Our study improves our understanding of how pneumococcal infection alters the lung environment, driving profound metabolic shifts that, in turn, influence bacterial phenotypes. This detailed understanding of host-pathogen metabolic interactions offers valuable insights into novel therapeutic strategies.

microbiology↗

bioRxiv2023

PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites

Streptococcus pneumoniae (Spn) resides in the nasopharynx where it can disseminate to cause disease. One key Spn virulence factor is pneumococcal surface protein A (PspA), which promotes survival by blocking the antimicrobial peptide lactoferricin. PspA has also been shown to mediate attachment to dying epithelial cells in the lower airway due to its binding of cell surface-bound mammalian (m)GAPDH. Importantly, the role of PspA during colonization is not well understood. Wildtype Spn was present in nasal lavage elutes collected from asymptomatically colonized mice at levels [~]10-fold higher that its isogenic PspA-deficient mutant ({Delta}pspA). Wildtype Spn also formed aggregates in mucosal secretions composed of sloughed epithelial cells and hundreds of pneumococci, whereas {Delta}pspA did not. Spn within the center of these aggregates better survived prolonged desiccation on fomites than individual pneumococci and were capable of infecting naive mice, indicating PspA-mediated aggregation conferred a survival/transmission advantage. Incubation of Spn in saline containing mGAPDH also enhanced tolerance to desiccation, but only for wildtype Spn. mGAPDH was sufficient to cause low-level aggregation of wildtype Spn but not {Delta}pspA. In strain WU2, the subdomain of PspA responsible for binding GAPDH (aa230-281) is ensconced within the lactoferrin (LF)-binding domain (aa167-288). We observed that LF inhibited GAPDH-mediated aggregation and desiccation tolerance. Using surface plasmon resonance, we determined that Spn forms multimeric complexes of PspA-GAPDH-LF on its surface and that LF dislodges GAPDH. Our findings have important implications regarding pneumococcal colonization/transmission processes and ongoing PspA-focused immunization efforts for this deadly pathogen. IMPORTANCEStreptococcus pneumoniae (Spn) is a dangerous human pathogen capable of causing pneumonia and invasive disease. The virulence factor pneumococcal surface protein A (PspA) has been studied for nearly four decades with well-established roles in pneumococcal evasion of C-reactive protein and neutralization of lactoferricin. Herein, we show that mammalian (m)GAPDH in mucosal secretions promotes aggregation of pneumococci in a PspA-dependent fashion, whereas lactoferrin counters this effect. PspA-mediated GAPDH-dependent bacterial aggregation protected Spn in nasal lavage elutes and grown in vitro from desiccation on fomites. Furthermore, surviving pneumococci within these aggregates retained their ability to colonize naive hosts after desiccation. We report that Spn binds to and forms protein complexes on its surface composed of PspA, mGAPDH, and lactoferrin. Changes in the levels of these proteins therefore most likely have critical implications on Spn colonization, survival on fomites, and transmission.

microbiology↗

bioRxiv2023

In vitro broad-spectrum antiviral activity of MIT-001, a mitochondria-targeted reactive oxygen species scavenger, against severe acute respiratory syndrome coronavirus 2 and multiple zoonotic viruses

The COVID-19 pandemic caused by SARS-CoV-2 becomes a serious threat to global health and requires the development of effective antiviral therapies. Current therapies that target viral proteins have limited efficacy with side effects. In this study, we investigated the antiviral activity of MIT-001, a small molecule reactive oxygen species (ROS) scavenger targeting mitochondria, against SARS-CoV-2 and other zoonotic viruses in vitro. The antiviral activity of MIT-001 was quantified by RT-qPCR and plaque assay. We also evaluated the functional analysis of MIT-001 by JC-1 staining to measure mitochondrial depolarization, total RNA sequencing to investigate gene expression changes, and immunoblot to quantify protein expression levels. The results showed that MIT-001 effectively inhibited the replication of B.1.617.2 and BA.1 strains, Zika virus, Seoul virus, and Vaccinia virus. Treatment with MIT-001 restored the expression of heme oxygenase-1 (HMOX1) and NAD(P)H: quinone oxidoreductase 1 (NqO1) genes, anti-oxidant enzymes reduced by SARS-CoV-2, to normal levels. The presence of MIT-001 also alleviated mitochondrial depolarization caused by SARS-CoV-2 infection. These findings highlight the potential of MIT-001 as a broad-spectrum antiviral compound that targets for zoonotic RNA and DNA viruses, providing a promising therapeutic approach to combat viral infection.

microbiology↗

bioRxiv2022

Engineered superinfective Pf phage prevents dissemination of Pseudomonas aeruginosa in a mouse burn model

Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa. Under stress conditions, mutations occurring in the Pf genome result in the emergence of super-infective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore SI-Pf can even infect Pf-lysogenized P. aeruginosa. Herein, we identified specific mutations located between the repressor and the excisionase genes that result in the emergence of SI-Pf. Based on these findings, we genetically engineered a SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening P. aeruginosa infection of burns caused by strain PAO1. eSI-Pf was able to infect PAO1 biofilms formed in vitro on polystyrene and inhibited their formation when at high concentration. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 hours. Importantly, and despite not lowering CFU/g of burn skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We determined that eSI-Pf induced a small colony variant of P. aeruginosa that was unable to disseminate systemically in our burned mouse model during acute infection. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial resistant P. aeruginosa infections of burn wounds. IMPORTANCEPseudomonas aeruginosa is a major cause of burn related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages have a set of drawbacks during phage therapy including the induction of bacterial resistance and limited bacteria-phage interactions in vivo. Here we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., using an engineered super-infective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.

microbiology↗

bioRxiv2022

Non-contact microfluidic analysis of the stiffness of single large extracellular vesicles from IDH1-mutated glioblastoma cells

In preparation for leveraging extracellular vesicles (EVs) for disease diagnostics and therapeutics, fundamental research is being done to understand EV biological, chemical, and physical properties. Most published studies investigate nanoscale EVs and focus on EV biochemical content. There is much less understanding of large microscale EV characteristics and EV mechanical properties. We recently introduced a non-contact microfluidic technique that measures the stiffness of large EVs (>1 m diameter). This study probes the sensitivity of the microfluidic technique to distinguish between EV populations by comparing stiffness distributions of large EVs derived from glioblastoma cell lines. EVs derived from cells expressing the IDH1 mutation, a common glioblastoma mutation known to disrupt lipid metabolism, were significantly stiffer than those expressed from wild-type cells. A supporting lipidomics analysis shows that the IDH1 mutation increases the amount of saturated lipids in EVs. Taken together, these data suggest that high-throughput microfluidics is capable of distinguishing between large EV populations that differ in biomolecular composition and therefore structure. These findings advance the understanding of EV biomechanics, in particular for the less studied microscale EVs, and demonstrate microfluidics to be a promising technique to perform clinical EV mechanophenotyping.

bioengineering↗

bioRxiv2021

Deep Learning-Based Phenotyping of Breast Cancer Cells Using Lens-free Digital In-line Holography

Lens-free digital in-line holography (LDIH) offers a wide field of view at micrometer-scale resolution, surpassing the capabilities of lens-based microscopes, making it a promising diagnostic tool for high-throughput cellular analysis. However, the complex nature of holograms renders them challenging for human interpretation, necessitating time- consuming computational processing to reconstruct object images. To address this, we present HoloNet, a novel deep learning architecture specifically designed for direct analysis of holographic images from LDIH in cellular phenotyping. HoloNet extracts both global features from diffraction patterns and local features from convolutional layers, achieving superior performance and interpretability compared to other deep learning methods. By leveraging raw holograms of breast cancer cells stained with well-known markers ER/PR and HER2, HoloNet demonstrates its effectiveness in classifying breast cancer cell types and quantifying molecular marker intensities. Furthermore, we introduce the feature-fusion HoloNet model, which extracts diffraction features associated with breast cancer cell types and their marker intensities. This hologram embedding approach allows for the identification of previously unknown subtypes of breast cancer cells, facilitating a comprehensive analysis of cell phenotype heterogeneity, leading to precise breast cancer diagnosis.

bioinformatics↗

bioRxiv2021

Capsule protects against intracellular killing and enables vascular endothelial cell translocation during invasive pneumococcal disease

Streptococcus pneumoniae (Spn) is a leading cause of invasive disease. Chief among its virulence determinants is capsular polysaccharide which protects the bacterium from phagocytosis. While 100 antigenically distinct capsule types are produced by Spn, i.e. serotypes, only 20-30 are commonly associated with invasive disease. A frequency that suggests serotypespecific properties of the capsule influence virulence. Herein, we show capsule has strong antioxidant properties. Moreover, that this property promotes invasive disease by protecting Spn taken up by vascular endothelial cells during bacteremia from endosome-killing and enhancing the translocation rate into organs. Crucially, isogenic capsule-switch mutants of Spn varied considerably in their resistance to H2O2-killing in culture and measured levels correlated positively with intracellular survival rates in vitro, organ invasion rates in vivo, and epidemiologically-established human attack rates for the corresponding serotype. The amount of capsule produced and specific biochemical features of a serotype, such as acetylation, also influenced Spn resistance to oxidative stress. Autolysin-mediated shedding was also found to be necessary, indicating that capsule worked as a distal sink for reactive oxygen species. Our results outline a new role for capsular polysaccharide, as an intracellular antioxidant. They help to explain why certain serotypes of Spn have greater propensity for human disease.

microbiology↗

bioRxiv2018

Bead Enhancement of EV Analysis

Extracellular vesicles (EVs) are recognized cancer biomarkers, however, clinical analysis has been difficult due to a lack of simple and sensitive assays. Here, we describe a bead-enhanced flow cytometry method, BEAD flow, using biotinylated EVs captured on streptavidin particles. With this method, we show analysis of patient-derived EVs using a panel of pancreatic cancer biomarkers. BEAD flow is easily translatable to any biomarker or cancer type and can be run with conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

cancer biology↗

bioRxiv2017

Deep-learning based hologram classification for molecular diagnostics

Lens-free digital in-line holography (LDIH) is a promising microscopic tool that overcomes several drawbacks (e.g., limited field of view) of traditional lens-based microcopy. However, extensive computation is required to reconstruct object images from the complex diffraction patterns produced by LDIH, which limits LDIH utility for point-of-care applications, particularly in resource limited settings. Here, we describe a deep transfer learning (DTL) based approach to process LDIH images in the context of cellular analyses. Specifically, we captured holograms of cells labeled with molecular-specific microbeads and trained neural networks to classify these holograms without reconstruction. Using raw holograms as input, the trained networks were able to classify individual cells according to the number of cell-bound microbeads. The DTL-based approach including a VGG19 pretrained network showed robust performance even with noisy experimental data. Combined with the developed DTL approach, LDIH could be realized as a low-cost, portable tool for point-of-care diagnostics.

bioengineering↗