Biology subjects

Pham, P.

Publications and source records attributed to Pham, P..

16 recordsLinked to original sources

bioRxiv2026

Astrocyte regulatory volume decrease is condition-dependent in intact brain tissue and requires the volume regulated anion channel

Multiple lines of evidence point to the volume regulated anion channel (VRAC) as being instrumental for cellular volume regulation in many cell types, including astrocytes. VRAC are thought to open during periods of astrocyte swelling, releasing anions and osmolytes to drive water out of the cell, allowing it to return to baseline volume even under sustained osmotic or ionic challenge, a process called regulatory volume decrease, or RVD. However, the occurrence of RVD and VRACs role in this process has remained controversial, with clear evidence in cultured cells but mixed reports from work in intact brain tissue. In the present study, we aimed to address this gap by generating a transgenic mouse line in which VRAC is conditionally ablated in astrocytes (VRAC cKO) and recording the volume responses of astrocytes in VRAC cKO and control tissue using real-time volume imaging. We found that the effect of VRAC cKO on astrocyte swelling was dependent on whether swelling was evoked by elevated extracellular potassium, or by reduced extracellular osmolarity. We also found that both VRAC and the presence of sufficient intracellular taurine concentration were required to elicit RVD in astrocytes, but only in hypoosmolar conditions. Our findings provide new information on the conditions needed to elicit RVD in intact brain tissue, and that VRAC is required for RVD to occur. Our findings further suggest that reduction of intracellular ion concentration is essential for VRAC to be activated, rather than simply membrane expansion. Future experiments will examine the solute release aspect of VRAC activation upon astrocyte swelling, as well as the contributions of VRAC to pathological volume dysregulation.

neuroscience↗

bioRxiv2026

Direct visualisation of post-replication gap formation at the bacterial RRS

Replication risk sequences (RRS) are recently discovered genomic structural elements that trigger post-replication gap formation during replisome passage. In E. coli, the two RRS elements are nearly perfect 222-bp G-quadruplex-containing repeats that flank the terminal domain and are highly conserved in both sequence and genomic position across enterobacteria. We report here the first direct visualisation of RRS function in vitro using single-molecule methods. When the G4 strand of the RRS element is positioned on the lagging-strand template, gaps are formed essentially every time a replisome encounters it. An increase in ssDNA in the synthesised DNA is readily seen using ssGAP-seq methods. When the G-quadruplex strand of the RRS is positioned on the leading-strand template, gaps are formed, albeit at lower frequency. However, the continued DNA synthesis in a rolling-circle assay indicates that the gaps are still formed on the lagging strand, indicating that the RRS complementary strand has a significant but reduced capacity to form a structure that triggers lesion skipping. The results document the potency of the RRS as a trigger for gap formation, suggesting a possible function for at least some eukaryotic G-quadruplexes.

biophysics↗

bioRxiv2025

EVOSYNTH: Enabling Multi-Target Drug Discovery through Latent Evolutionary Optimization and Synthesis-Aware Prioritization

Complex diseases, such as cancer and neurodegeneration, feature interconnected pathways, making single-target therapies ineffective due to pathway redundancy and compensatory mechanisms. Polypharmacy, which combines multiple drugs to target distinct proteins, addresses this but often leads to drug-drug interactions, cumulative toxicity, and complex pharmacokinetics. To overcome these challenges, we introduce EO_SCPLOWVOC_SCPLOWSO_SCPLOWYNTHC_SCPLOW, a modular framework for multi-target drug discovery that combines latent evolution and synthesis-aware prioritization to generate and prioritize candidates with high translational potential. Latent evolution navigates a chemically and functionally informed latent space to identify candidates with strong predicted affinity across multiple targets. Synthesis-aware prioritization evaluates both retrosynthetic feasibility and the trade-off between synthetic cost and therapeutic reward, enabling practical and efficient candidate selection. Applied to dual inhibition of JNK3 and GSK3{beta} in Alzheimers disease and PI3K and PARP1 in ovarian cancer, EO_SCPLOWVOC_SCPLOWSO_SCPLOWYNTHC_SCPLOW consistently outperforms baseline generative models, achieving higher predicted affinities, improved scaffold diversity, and lower synthesis costs. These findings highlight EO_SCPLOWVOC_SCPLOWSO_SCPLOWYNTHC_SCPLOWs ability to integrate target-driven generation with practical synthesizability, establishing a scalable framework for multi-target and polypharmacological drug discovery. Our source code and data to reproduce all experiments is publicly available on GitHub at: https://github.com/HySonLab/EvoSynth.

bioinformatics↗

bioRxiv2025

Distinct Neurogenic Dynamics of Cortico-cortical Neuronal Subtypes in Layer 2/3 of the Mouse Visual Cortex

In the mammalian cerebral cortex, the birthdates of excitatory projection neurons are closely linked to their laminar positions, which are often associated with distinct long-range projection targets. Although broad relationships between neurogenic timing, laminar position, and projection patterns are well established, the degree to which birthdate specifies projection identity within the same cortical layer remains unclear. In the mouse primary visual cortex (V1), neurons projecting to lateral higher visual areas are relatively evenly distributed throughout layer 2/3, whereas those projecting to medial areas are biased toward more superficial sublayers. To determine whether these projection identities are linked to neurogenic timing, we combined EdU birthdating with retrograde viral tracing. Notably, we found that V1 layer 2/3 neurons projecting to lateral higher visual areas are preferentially born at embryonic day 15.5 (E15.5) compared to E16.5, whereas V1 neurons projecting to medial higher visual areas show no significant birthdate bias between E15.5 and E16.5. These findings suggest that distinct cortico-cortical projection subtypes in layer 2/3 are generated on different temporal schedules, linking neurogenic timing to fine-scale projection identity.

neuroscience↗

bioRxiv2025

LANTERN: Leveraging Large Language Models and Transformers for Enhanced Molecular Interactions

Understanding molecular interactions such as Drug-Target Interaction (DTI), Protein-Protein Interaction (PPI), and Drug-Drug Interaction (DDI) is critical for advancing drug discovery and systems biology. However, existing methods often struggle with scalability due to the vast chemical and biological space and suffer from limited accuracy when capturing intricate biochemical relationships. To address these challenges, we introduce LANTERN (Leveraging Large LANguage Models and Transformers for Enhanced moleculaR interactioNs), a novel deep learning framework that integrates Large Language Models (LLMs) with Transformer-based architectures to model molecular interactions more effectively. LANTERN generates high-quality, context-aware embeddings for drug and protein sequences, enabling richer feature representations and improving predictive accuracy. By leveraging a Transformer-based fusion mechanism, our framework enhances scalability by efficiently integrating diverse interaction data while maintaining computational feasibility. Experimental results demonstrate that LANTERN achieves state-of-the-art performance on multiple DTI and DDI benchmarks, significantly outperforming traditional deep learning approaches. Additionally, LANTERN exhibits competitive performance on challenging PPI tasks, underscoring its versatility across diverse molecular interaction domains. The proposed framework offers a robust and adaptable solution for modeling molecular interactions, efficiently handling a diverse range of molecular entities without the need for 3D structural data and making it a promising framework for foundation models in molecular interaction. Our findings highlight the transformative potential of combining LLM-based embeddings with Transformer architectures, setting a new standard for molecular interaction prediction. The source code and relevant documentation are available at: https://github.com/HySonLab/LANTERN.

bioinformatics↗

bioRxiv2024

The RNA-binding protein HuR impairs adipose tissue anabolism in pancreatic cancer cachexia

BackgroundCachexia is defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC), and negatively impacts patient outcomes. Nutritional supplementation cannot fully reverse tissue wasting, and the mechanisms underlying this phenotype are unclear. This work aims to define the relative contributions of catabolism and anabolism to adipose wasting in PDAC-bearing mice. Human antigen R (HuR) is an RNA-binding protein recently shown to suppress adipogenesis. We hypothesize that fat wasting results from a loss of adipose anabolism driven by increased HuR activity in adipocytes of PDAC-bearing mice. MethodsAdult C57BL/6J mice received orthotopic PDAC cell (KrasG12D; p53R172H/+; Pdx1-cre) (PDAC) or PBS (sham) injections. Mice exhibiting moderate cachexia (9 days after injection) were fasted for 24h, or fasted 24h and refed 24h before euthanasia. A separate cohort of PDAC mice were treated with an established HuR inhibitor (KH-3, 100 mg/kg) and subjected to the fast/refeed paradigm. We analyzed body mass, gross fat pad mass, and adipose tissue mRNA expression. We quantified lipolytic rate as the normalized quantity of glycerol released from 3T3-L1 adipocytes in vitro, and gonadal fat pads (gWAT) ex vivo. Results3T3-L1 adipocytes treated with PDAC cell conditioned media (CM) had lower expression of lipolysis and lipogenesis genes than control cells, and did not display elevated lipolysis as measured by liberated glycerol. PDAC gWAT cultured ex vivo displayed decreased lipolysis compared to sham gWAT (-54.7%). PDAC and sham mice lost equivalent fat mass after a 24h fast, however, PDAC mice could not restore inguinal fat pads (iWAT) (-40.5%) or gWAT (-31.8%) mass after refeeding. RNAseq revealed 572 differentially expressed genes in gWAT from PDAC compared to sham mice. Downregulated genes (n=126) were associated with adipogenesis (adj p=0.05), and expression of adipogenesis master regulators Pparg and Cebpa were reduced in gWAT from PDAC mice. Immunohistochemistry revealed increased HuR staining in gWAT (+74.9%) and iWAT (+41.2%) from PDAC mice. Inhibiting HuR binding restored lipogenesis in refed animals with a concomitant increase in iWAT mass (+131.7%). ConclusionsOur work highlights deficient adipose anabolism as a driver of reduced lipid content in 3T3-L1 adipocytes treated with PDAC conditioned media and PDAC mice. The small molecule KH-3, which disrupts HuR binding, restored adipose anabolism in PDAC mice. This highlights HuR as a potentially targetable regulatory node for adipose anabolism in cancer cachexia.

cancer biology↗

bioRxiv2024

Generative AI-assisted Virtual Screening Pipeline for Generalizable and Efficient Drug Repurposing

Drug repurposing presents a valuable strategy to expedite drug discovery by identifying new therapeutic uses for existing compounds, especially for diseases with limited treatment options. We propose a Generative AI-assisted Virtual Screening Pipeline that combines generative modeling, binding pocket prediction, and similarity-based searches within drug databases to achieve a generalizable and efficient approach to drug repurposing. Our pipeline enables blind screening of any protein target without requiring prior structural or functional knowledge, allowing it to adapt to a wide range of diseases, including emerging health threats and novel targets where information is scarce. By rapidly generating potential ligands and efficiently identifying and ranking drug candidates, our approach accelerates the drug discovery process, broadening the scope and impact of repurposing efforts and offering new possibilities for therapeutic development. Detailed results and implementation can be accessed at https://github.com/HySonLab/DrugPipe.

bioinformatics↗

bioRxiv2023

A foundational atlas of autism protein interactions reveals molecular convergence

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

systems biology↗

bioRxiv2023

Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex

Glioblastoma (GBM) represents the most aggressive subtype of glioma, noted for its profound invasiveness and molecular heterogeneity. The mesenchymal (MES) transcriptomic subtype is frequently associated with therapy resistance, rapid recurrence, and increased tumor-associated macrophages. Notably, activation of the NF-{kappa}B pathway and alterations in the PTEN gene are both associated with this malignant transition. Although PTEN aberrations have been shown to be associated with enhanced NF-{kappa}B signaling, the relationships between PTEN, NF-{kappa}B and MES transition are poorly understood in GBM. Here, we show that PTEN regulates the chromatin binding of bromodomain and extraterminal (BET) family proteins, BRD2 and BRD4, mediated by p65/RelA localization to the chromatin. By utilizing patient-derived glioblastoma stem cells and CRISPR gene editing of the RELA gene, we demonstrate a crucial role for RelA lysine 310 acetylation in recruiting BET proteins to chromatin for MES gene expression and GBM cell invasion upon PTEN loss. Remarkably, we found that BRD2 is dependent on chromatin associated acetylated RelA for its recruitment to MES gene promoters and their expression. Furthermore, loss of BRD2 results in the loss of MES signature, accompanied by an enrichment of proneural signature and enhanced therapy responsiveness. Finally, we demonstrate that disrupting the NF-{kappa}B/BRD2 interaction with a brain penetrant BET-BD2 inhibitor reduces mesenchymal gene expression, GBM invasion, and therapy resistance in GBM models. This study uncovers the role of hitherto unexplored PTEN-NF-{kappa}B-BRD2 pathway in promoting MES transition and suggests inhibiting this complex with BET-BD2 specific inhibitors as a therapeutic approach to target the MES phenotype in GBM.

cancer biology↗

bioRxiv2023

The great divide: rhamnolipids mediate separation between P. aeruginosa and S. aureus

The coexistence of multiple bacterial species during infection can have significant impacts on pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that can co-infect hosts and cause serious illness. The factors that dictate whether one species will outcompete the other or whether the two species can coexist are not fully understood. We investigated the role of surfactants in the interactions between these two species on a surface that enables P. aeruginosa to swarm. We found that P. aeruginosa swarms are repelled by colonies of clinical S. aureus isolates, creating physical separation between the two strains. This effect was abolished in mutants of S. aureus that were defective in the production of phenol-soluble modulins (PSMs), which form amyloid fibrils around wild-type colonies. We investigated the mechanism that establishes physical separation between the two species using the Imaging of Reflected Illuminated Structures (IRIS) method, which tracks the flow of the rhamnolipid surfactant layer produced by P. aeruginosa. We found that PSMs produced by S. aureus deflected the rhamnolipid surfactant layer flow, which in turn, altered the direction of P. aeruginosa swarms. These findings show that rhamnolipids mediate physical separation between P. aeruginosa and S. aureus, which enables these species to coexist in distinct microenvironments. Additionally, we found that a Bacillus subtilis surfactant and abiotic hydrophobic molecules repelled P. aeruginosa swarms through surfactant deflection. Our results suggest that surfactant interactions could have major impacts on bacteria-bacteria and bacteria-host relationships. In addition, our findings uncover a mechanism responsible for P. aeruginosa swarm development that does not rely on sensing but instead is guided largely by the flow of the surfactant layer and its boundaries.

microbiology↗

bioRxiv2023

Assessment of three-dimensional RNA structure prediction in CASP15

The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report assessments of RNA structure predictions in CASP15, the first CASP exercise that involved RNA structure modeling. Forty two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, which did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and X-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global fold of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as non- canonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.

biophysics↗

bioRxiv2023

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 4,,12,i:- ST34 in Southeast Asia

Salmonella enterica serotype 4,[5],12,i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogens epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a hotspot for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors. Our work enriches the understanding on epidemiology and evolution of this variant in Southeast Asia, and determines that multidrug resistance plasmids have driven its local and potentially global success.

genomics↗

bioRxiv2022

RNA structure landscape of S. cerevisiae introns

Pre-mRNA secondary structures are hypothesized to play widespread roles in regulating RNA processing pathways, but these structures have been difficult to visualize in vivo. Here, we characterize S. cerevisiae pre-mRNA structures through transcriptome-wide dimethyl sulfate (DMS) probing, enriching for low-abundance pre-mRNA through splicing inhibition. We cross-validate structures found from phylogenetic and mutational studies and identify new structures within the majority of probed introns (102 of 161). We find widespread formation of "zipper stems" between the 5 splice site and branch point, "downstream stems" between the branch point and the 3 splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals examples where intron structures can form in vitro without the presence of binding partners, and structure ensemble prediction suggests that such structures appear in introns across the Saccharomyces genus. We develop a high-throughput functional assay to characterize variants of RNA structure (VARS-seq) and we apply the method on 135 sets of stems across 7 introns, identifying structured elements that alter retained intron levels at a distance from canonical splice sites. This transcriptome-wide inference of intron RNA structures suggests new ideas and model systems for understanding how pre-mRNA folding influences gene expression.

biochemistry↗

bioRxiv2021

Colicin-mediated transport of DNA through the iron transporter FepA

Colicins are protein antibiotics used by bacteria to eliminate competing Escherichia coli. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate through the strictly impermeable bacterial cellular envelope. Here, applying live-cell fluorescence imaging we were able to follow colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB is also transported into the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA, applying a combination of photo-activated crosslinking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with a large-scale conformational change in the colicin. Finally In vivo crosslinking experiments and supplementary simulations of the translocation process indicate that part of the colicin engages active transport by disguising itself to part of the cellular receptor.

microbiology↗

bioRxiv2020

Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations

Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1 infected patients in the absence of mutations in the proteins targeted by these drugs. We previously reported that, in vitro, the lab-adapted NL4-3 strain of HIV-1 can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance the ability of HIV-1 to spread via cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can broadly confer resistance to multiple classes of ARVs in the context of cell-cell but not cell-free infection and also increase resistance to ARVs when coupled with target-gene drug resistance mutations. To investigate the mechanism of Env-mediated drug resistance, we evaluated the impact of the Env mutations on Env stability and conformational dynamics. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation compared to WT virus and reduced rates of gp120 shedding. We also selected for mutations in the gp41 ectodomain of clinically relevant, CCR5-tropic isolates in the presence of DTG. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication kinetics and Env structure that are HIV-1 strain-dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest a "stepping-stone" model whereby mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV-target genes. Author summaryAlthough combination antiretroviral (ARV) therapy has proven highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle to long-term treatment, particularly in resource-limited settings. In most cases, resistance arises from the accumulation of mutations in the ARV-target genes; however, in some cases, resistance develops without ARV target-gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo. We demonstrate that Env mutations can confer broad resistance to multiple classes of ARVs and define the effect of the Env mutations on Env subunit interactions and sensitivity to neutralizing antibodies. We also selected for drug resistance mutations in Env in clinically relevant HIV-1 isolates. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based, Env-mediated drug resistance may impact current and possibly future therapeutic strategies. Our findings also provide clues towards understanding how ARV-treated patients can experience virological failure without acquiring drug resistance mutations in ARV-target genes.

microbiology↗

bioRxiv2018

Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). We apply the phasor-FLIM method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify the developmental, or \"D-trajectory\", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined an Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis, a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. This may increase embryo implantation success for in vitro fertilization clinics.\n\nHighlightsO_LIA label-free method of tracking metabolic trajectories during pre-implantation mouse embryo development.\nC_LIO_LIA non-invasive approach for assessing embryo quality and viability by a phasor-FLIM analysis.\nC_LI

developmental biology↗