Biology subjects

Nguyen, T. P.

Publications and source records attributed to Nguyen, T. P..

13 recordsLinked to original sources

bioRxiv2026

Systematic identification of seed-driven off-target effects in Perturb-seq experiments

Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.

genomics↗

bioRxiv2026

Transient contractility attenuation reprograms epithelial cells into a protrusion-driven state that drives tissue fluidization

Collective cell migration drives tissue morphogenesis, repair and remodeling, and is often accompanied by transitions from solid-like to fluid-like states. While such tissue fluidization has been linked to physical parameters such as cell density, shape and activity, how it is actively regulated by mechano-chemical interplay remains unclear. Previous research has shown that transient attenuation of actomyosin contractility induces a transition from pulsatile, spatially confined motion to coherent, persistent long-range collective flow; however, the underlying cellular and signaling mechanisms remain unclear. Here we uncover the mechanistic basis by which transient perturbation of cell contractility reprograms the migration mode of confluent epithelial cells into a protrusion-driven, fluidizing state, by combining kinase-reporter live imaging, force measurements and mathematical modeling. This transition arises from coordinated changes in cell morphology and mechanics, including reduced cortical tension and greater stretch-induced cell strain, together with enhanced cell-substrate adhesion and force transmission. At the signaling level, this process is accompanied by a rewiring of extracellular signal-regulated kinase (ERK)-mediated mechanotransduction toward a protrusion-coupled mode that sustains migration even under fully confluent conditions. Consistently, a multicellular computational model further demonstrates that protrusion-driven migration is sufficient to promote shape-velocity alignment and drive a transition from caged to flocking-like collective states. Together, our results identify transient contractility attenuation followed by recovery as a trigger for a protrusion-driven state that fluidizes confluent epithelial tissues through coordinated remodeling of cytoskeletal, adhesive, and signaling systems.

synthetic biology↗

bioRxiv2025

Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites

Lysosomal dysfunction is a well-recognized feature of aging, yet its systematic molecular investigation remains limited. Here, we employ a suite of tools for rapid lysosomal isolation to construct a multi-tissue atlas of the metabolite changes that murine lysosomes undergo during aging. Aged lysosomes in brain, heart, muscle and adipose accumulate glycerophosphodiesters and cystine, metabolites that are causally linked to juvenile lysosomal storage disorders like Batten disease. Levels of these metabolites increase linearly with age, preceding organismal decline. Caloric restriction, a lifespan-extending intervention, mitigates these changes in the heart but not the brain. Our findings link lysosomal storage disorders to aging-related dysfunction, uncover a metabolic lysosomal "aging clock," and open avenues for the mechanistic investigation of how lysosomal functions deteriorate during aging and in age-associated diseases. One-Sentence SummaryAging in mice is tracked by a lysosomal "clock", where glycerophosphodiesters and cystine - metabolites causally linked to juvenile lysosomal storage disorders - gradually accumulate in lysosomes of the brain, heart, skeletal muscle and adipose tissue.

cell biology↗

bioRxiv2025

An emergent disease-associated motor neuron state precedes cell death in a mouse model of ALS

To uncover molecular determinants of motor neuron degeneration and selective vulnerability in amyotrophic lateral sclerosis (ALS), we generated longitudinal single-nucleus transcriptomes and chromatin accessibility profiles of spinal motor neurons from the SOD1-G93A ALS mouse model. Vulnerable alpha motor neurons showed thousands of molecular changes, marking a transition into a novel cell state we named disease-associated motor neurons (DAMNs). We identified transcription factor regulatory networks that govern how healthy cells transition into DAMNs as well as those linked to vulnerable and resistant motor neuron subtypes. Using spatial transcriptomics, we found reactive glia located near motor neurons early in disease, suggesting early signaling events between motor neurons and glia. Finally, we found that the human orthologs of genomic regions with differential accessibility in SOD1-G93A alpha motor neurons are enriched for single nucleotide polymorphisms associated with human ALS, providing evidence that the genetic underpinnings of motor neuron vulnerability are conserved.

neuroscience↗

bioRxiv2025

Chalcogen derivatives for the treatment of African trypanosomiasis: biological evaluation of thio and seleno- semicarbazones and their azole derivatives

Human African Trypanosomiasis (HAT) is caused by Trypanosoma brucei. Drug therapy remains challenging due to drug resistance and/or toxicity. New drugs are needed. Using thiosemicarbazones as a starting point, we employed a S to Se isosteric replacement strategy to design 44 analogs which were evaluated against T. brucei in vitro. Compounds were divided into eleven groups of four derivatives corresponding to thio-, selenosemicarbazones, and their cyclic counterparts, thio- and selenazoles. We selected three groups which contained a total of six derivatives that inhibited parasite growth by >70%. Then, we investigated the mechanism of action of these compounds, performing quantitative assays to measure their inhibition of the T. brucei cathepsin L-like protease (TbrCATL) and DPPH antioxidant activities. The lead compound (SeO3) showed antioxidant capacity and the best activity against T. brucei (EC50 = 0.47 {micro}M). Nevertheless, its toxicity should be improved. We also predicted the interactions of these compounds with TbrCATL utilizing molecular dynamics. We demonstrate that the Se derivatives are more active than their S analogues, and that the selenazole ring decreases Se-associated toxicity. Also, thio- and selenosemicarbazones are more potent against TbrCATL than the cyclic derivatives. We conclude that TbrCATL inhibition should be combined with antioxidant activity to obtain active compounds against T. brucei.

microbiology↗

bioRxiv2025

Competitive elimination of ZO-1/ZO-2-deficient cells regulates epithelial barrier homeostasis

Epithelia cover the body and form a barrier to segregate the internal body from the external environment. Epithelial tissues contact the external environment and are exposed to various stresses that are potentially deleterious for the epithelial barrier1. However, how focal epithelial barrier defects are detected and repaired to maintain epithelial barrier homeostasis remains poorly understood. We co-cultured ZO-1/ZO-2 double-knockout (DKO) MDCK II cells, which lack tight junctions, with wild-type MDCK II cells, to understand how epithelial cells respond to focal epithelial barrier defects. When co-cultured with wild-type cells, ZO-1/ZO-2 DKO cells were selectively eliminated by induction of apoptosis. The elimination depended on a purse-string-like contraction of supracellular actomyosin cables formed at the clone boundary, regulated by ROCK. Furthermore, Hippo signaling and adherens junctions in the surrounding wild-type cells were required to eliminate ZO-1/ZO-2 DKO cells. These results demonstrate that selective elimination of ZO-1/ZO-2-deficient cells by cell competition regulates epithelial barrier homeostasis. In briefOtani et al. reveal that epithelial barrier homeostasis is regulated by cell competition-mediated elimination of ZO-1/ZO-2-deficient cells. Supracellular actomyosin cables form at the clone boundary and constrict in a purse-string-like manner to eliminate the loser cells. Mechanosensing in the winning cells is required to eliminate the loser cells. HighlightsO_LIZO-1/ZO-2-deficient cells are eliminated by cell competition C_LIO_LIActomyosin cables form in the winning cells at the clone boundary C_LIO_LIA purse-string-like contraction of actomyosin cables compresses the losing cells C_LIO_LIMechanosensing in the winning cells is important to eliminate the loser cells C_LI

cell biology↗

bioRxiv2024

Characterization of Autophagy Cargo Profiles during Starvation and Exercise with a Novel Autophagosome Isolation Method

Autophagy is a cellular process to clear unwanted and dysfunctional cellular cargoes, which are sequestered in autophagosomes before their delivery to lysosomes for degradation. Autophagy cargo selection, mediated by cargo receptors, varies across cell types and conditions. Understanding the cargo features is essential for elucidating autophagys function in specific physiological or pathological contexts. Here we present a simple and rapid method for isolating LC3B-positive autophagosomes from the tissues of GFP-LC3 transgenic mice, a widely used autophagy reporter model, without relying on the complex ultracentrifugation steps required by traditional methods. When combined with quantitative proteomics, this approach enables efficient in vivo characterization of autophagy cargoes. We applied this method to establish autophagy cargo profiles in skeletal muscle during starvation and exercise, two physiological conditions that activate autophagy, and identified distinct cargo selection patterns, with significantly higher levels of ER-phagy and ribophagy observed during starvation. We further revealed the ER-phagy receptors TEX264 and RETREG1/FAM134B as potential mediators of the elevated ER-phagy under starvation. In summary, we report an efficient workflow for in vivo autophagy cargo characterization and provide detailed analysis and comparison of cargo profiles under starvation and exercise conditions.

cell biology↗

bioRxiv2024

An Enzyme-Linked Immunosorbent Assay (ELISA)-based Activity Assay for AMP-Activated Protein Kinase (AMPK)

AMP-activated protein kinase (AMPK) is the master regulator of cellular and organismal energy homeostasis, playing an essential role in modulating metabolism as well as other cellular processes. Efforts have been made to discover pharmacological modulators of AMPK activity because of their therapeutic potentials against various diseases. Measuring AMPK activity in vitro is a fundamental step for testing AMPK activators and inhibitors. Here we report an enzyme-linked immunosorbent assay (ELISA)-based AMPK activity assay with simple steps and high sensitivity, which represents an alternative in-house method to the traditional radioactive method as well as other techniques that require special reagents and commercial reagent kits.

biochemistry↗

bioRxiv2023

Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions undergo spontaneous fracture when claudin/JAM-A-deficient cells are exposed to mechanical stress. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells. SummaryTight junction membrane proteins claudins, JAM, and CAR coordinately regulate the nanometer-scale organization of ZO-1 molecules, and are required for the mechanical resistance of apical junctions in epithelial cells.

cell biology↗

bioRxiv2023

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

biochemistry↗

bioRxiv2022

CLIP-Seq Analysis Enables the Design of Ribosomal RNA Bait Oligonucleotides That Protect Against C9orf72 ALS/FTD-Associated Poly-GR Pathophysiology

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind to low complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remains unclear. We used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits in cells and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal disfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

neuroscience↗

bioRxiv2020

Distinct VIP interneurons in the cingulate cortex encode anxiogenic and social stimuli

A hallmark of higher-order cortical regions is their functional heterogeneity, but it is not well understood how these areas encode such diverse information. The anterior cingulate cortex (ACC), for example, is important in both emotional regulation and social cognition. Previous work shows activation of the ACC to anxiety-related and social stimuli, but it is unknown how subpopulations or microcircuits within the ACC simultaneously encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to alter the activity of many cells in local cortical microcircuits, but it is unknown whether the activity of VIP cells in the ACC (VIPACC) encodes anxiety-related or social information. Using in vivo calcium imaging and miniscopes in freely behaving mice to monitor VIPACC activity, we identified distinct, non-overlapping subpopulations of VIPACC that preferentially activated to either anxiogenic, anxiolytic, social, or non-social stimuli. We determined that stimulus-selective cells encode the animals behavioral states and VIP interneuron clusters may co-activate, improving this encoding. Finally, we used trans-synaptic tracing to show that VIPACC receive widespread inputs from regions implicated in emotional regulation and social cognition. These findings demonstrate not only that the ACC is not homogeneous in its function, but also that there is marked functional heterogeneity even within disinhibitory interneuron populations. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

neuroscience↗

bioRxiv2018

Phase of neuronal activity encodes 2-dimensional space in the human entorhinal cortex

The entorhinal cortex plays a vital role in our spatial awareness. Much focus has been placed on the spatial activity of its individual neurons, which fire in a grid-like pattern across an environment1. On a population level, however, neurons in the entorhinal cortex also display coherent rhythmic activity known as local field potential. These local field oscillations have been shown to correlate with behavioural states but it remains unclear how these oscillations relate to spatial behaviour and the spatial firing pattern of individual neurons. To investigate this, we recorded entorhinal cortical neurons in the human brain during spatial memory tasks performed in virtual environments. We observed a spatial modulation of the phase of action potentials relative to the local field potentials. In addition, the spike phase modulation displayed correlation with the movement of the avatar, displayed discrete phase tuning at the cellular level, rotated phase between electrodes, and expressed spatially coherent phase maps that scaled with the virtual environment. Using surrogate data, we demonstrated that spike phase coherence is dependent on the spatial phase dynamics of gamma oscillations. We argue that the spatial coordination of spike generation with gamma rhythm underlies the emergence of grid cell activity in the entorhinal cortex. These results shed a new light on the intricate interlacing between the spiking activity of neurons and local field oscillations in the brain.

neuroscience↗