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Lim, A.

Publications and source records attributed to Lim, A..

At least 19 recordsLinked to original sources
bioRxiv2026

Stereociliary localization and function of the tip-link protein PCDH15-CD2 are regulated by the PDZ-scaffold NHERF-1

Sensory hair cells in the inner ear convert mechanical sound deflections into electrical signals via the stereociliary hair bundle on their apical surface. Proper bundle function requires that structural, adhesion, and motor proteins be delivered to specific stereocilia sub-compartments and maintained at precise levels throughout life, yet the underlying mechanisms remain poorly understood. Here we investigated how spatiotemporal localization of Protocadherin-15 (PCDH15), a component of kinociliary links, transient lateral links, and tip links, is regulated in mouse auditory hair cells. Using biochemical assays in vitro, we identified the PDZ-domain scaffolds NHERF-1/2 as direct binding partners of CD2, a PCDH15 isoform essential for hearing. We mapped the interaction to NHERF-1's PDZ1 domain and CD2's PDZ-binding motif (PBM), and showed that this binding is regulated by phosphorylation of the CD2 PBM. Using Nherf1 knockout and Pcdh15-CD2 knock-in mice defective in this interaction, we found CD2-NHERF-1 binding is required for proper CD2 levels and localization in the developing hair bundle. Loss of this interaction largely spared early bundle morphogenesis and MET function but caused high-frequency hearing loss and degeneration of the shortest stereociliary row in adult mice. Together, these findings define an important regulatory mechanism with therapeutic relevance for hair bundle repair.

cell biology↗
bioRxiv2026

Systematic benchmarking of multi-modal approaches for tumor-naive ctDNA detection and quantification

Longitudinal monitoring of circulating tumor DNA (ctDNA) has emerged as a promising framework for characterizing treatment response dynamics in cancer. Scalable tumor-naive approaches for quantifying ctDNA often involve whole-genome sequencing (WGS) or DNA methylation profiling, but their comparative performance and capacity for complementary integration remain poorly understood. Here we systematically benchmarked tumor-naive WGS- and methylation-based ctDNA quantification methods using plasma from 150 patients with colorectal, lung and breast cancer. Using paired high-depth WGS and EM-seq data, we generated 40,000 in silico samples and evaluated detection accuracy, limits of detection (LoD) and quantification (LoQ) across cancer types and sequencing depths (0.1x-30x). We further assessed single- and multimodal method combinations, identifying conditions under which integrated approaches enhance analytical performance for detection and quantification relative to single modalities. This benchmark delineates key performance trade-offs and provides a practical framework to support method development and guide future research applications in ctDNA-based biomarker studies.

bioinformatics↗
bioRxiv2026

Deep Learning-Guided Holotomography Reveals Early Structural Remodelling During Pluripotency Exit

Real-time assessment of human pluripotent stem cell (hPSC) quality is critical for reproducibility and safety in regenerative medicine, yet current methods are invasive, labor-intensive, or highly operator-dependent. We present DeepHOPE (Deep-learning-guided Holotomography for Pluripotency Evaluation), a non-invasive, automatizable, and statistics-driven platform that integrates three-dimensional (3D) refractive index imaging with deep learning to assess pluripotency. DeepHOPE performs robustly across diverse contexts, including germ-layer differentiation, retinoic acid-induced differentiation, and mid-reprogramming cultures, enabling streamlined cell production workflows and improving the efficiency of midbrain dopaminergic neuron differentiation through informed colony selection. Mechanistically, DeepHOPE detects minute colony-scale topological changes that precede molecular loss of pluripotency. These early changes are associated with rapid F-actin remodeling, including apical-to-basal redistribution during early differentiation. Consistent with a functional role for cytoskeletal regulation in state transitions, sustained reduction of actomyosin tension decreases pluripotency, identifying cytoskeletal dynamics as an upstream determinant of early pluripotency exit. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/720508v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1d73508org.highwire.dtl.DTLVardef@1dab29aorg.highwire.dtl.DTLVardef@1039f76org.highwire.dtl.DTLVardef@da29a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗
bioRxiv2026

Resolving the Activation Mechanism of the Human 20S Proteasome

Proteasome activators (PAs) bind the -subunits of the human 20S proteasome (h20S), opening the "gates" and allowing entry of substrate proteins. Aging is associated with diminished proteasome activity, leading to interest in understanding this activation mechanism. Evolving models have been proposed regarding PAs C-terminal tails, yet the critical molecular contacts for gate-opening are unclear. Here, we show a conserved leucine in the 5th position (P5) of the C-terminus is essential for h20S gate opening. By engineering C-termini in a model activator, PA26E102A, we show mutations to P5 systematically modulate proteasome activity in vitro and in cells. Structures of PA26E102A:h20S complexes at 2.7-3.2 [A] resolution identify interactions between P5 and a conserved arginine in the h20S, leading to partial or full gate opening. These results clarify the essential contacts required for h20S gate opening, potentially enabling design of proteasome activation therapies.

biophysics↗
bioRxiv2026

A Multispecies, Modality-Agnostic Scalable In Vivo Mosaic Screening Platform for Therapeutic Target Discovery

Validating therapeutic targets for complex diseases requires investigating gene functions within native tissue architectures rather than reductionist in vitro models. Here we present a modality-agnostic AAV-based in vivo high-throughput screening platform capable of delivering knockouts, gain-of-function, and synthetic miRNA knockdowns directly to cells within the diseased environment. This system scales to hundreds of perturbations and is adaptable to diverse species and organ systems. To translate high-dimensional screen data into therapeutic assessment, we established a curated analysis framework that scores single-cell transcriptomes against human disease-specific molecular signatures. This method enables quantitative ranking of targets across distinct biological domains ranging from structural fibrosis to inflammatory signaling, to narrow in on the therapeutic potential of each intervention. We applied this strategy to screen loss- and gain-of-function libraries in a murine pulmonary fibrosis model and within the spontaneously osteoarthritic joints of aged horses, identifying metabolic, antifibrotic, and immunomodulatory targets. Importantly, our analysis framework successfully predicted functional outcomes in orthogonal human ex vivo tissue models, including soluble collagen reduction in lung slices and glycosaminoglycan restoration in cartilage, thus establishing a powerful paradigm for prioritizing therapeutic targets by uniting human disease signatures with highly multiplexed in vivo functional genomics.

systems biology↗
bioRxiv2026

Automation of Complex Patient-Derived Organoid (PDO) Culture and Screen

Tissue organoids and spheroids are powerful tools for many different applications, particularly in the area of drug screening. Yet, their widespread use is limited by a need for automated cell and tissue culture procedures and increased throughput. Here, we demonstrate that the BioAssembyBot(R) (BAB) automation platform, with integrated BioStorageBot(R) (BSB) and imaging system can perform tasks normally done by scientists with increased efficiency and reduced error. Using the BAB Hand(R)| Pipette tool, BAB can precisely seed a Matrigel(R) organoid suspension such that Matrigel(R) domes are formed consistently in the center of each well plate. The BAB-automated process performed faster, with a higher success rate, and less organoid fragmentation than when the process is done by an experienced scientist. We then repeated this workflow with patient-derived colorectal cancer organoids (CRC PDOs) and performed a fully automated drug screen on cultured organoids. Image-based profiling performed on images that were automatically acquired was able to distinguish clear differences in patient response to anticancer drugs between two donors. Overall, we demonstrate the capability of the BioAssemblyBot(R) automation platform to manage an entire organoid drug screening protocol with minimal human interaction. The system is user-friendly and may be adapted to a wide variety of workflows and applications, providing a solution to the need for organoid automation technology.

cancer biology↗
bioRxiv2025

Single-cell atlas of the developing Down syndrome brain cortex

Down syndrome (DS), caused by trisomy of chromosome 21, is the leading genetic cause of intellectual disability, yet the mechanisms disrupting fetal brain development remain unclear. We performed single-cell transcriptomic and chromatin accessibility profiling of approximately 250,000 cells from 15 DS and 15 control human fetal cortices (10-20 weeks post-conception). Our analysis revealed a subtype-specific reduction in RORB/FOXP1-expressing excitatory neurons and widespread disruption of neurodevelopmental transcriptional programs. Chromosome 21 transcription factors BACH1, PKNOX1, and GABPA emerged as dosage-sensitive hubs regulating genes linked to intellectual disability. Antisense oligonucleotide-mediated normalization of these factors in human neural progenitors in vitro partially rescued target gene expression. Benchmarking a humanized in vivo model captured additional molecular and cellular signatures of DS, complementing the in vitro model. Together, this resource defines the gene-regulatory landscape underlying cortical development in DS and highlights candidate molecular targets and preclinical models for future intervention studies. HighlightsO_LISingle-cell atlas of Down syndrome fetal cortex links transcriptional dysregulation to reduction of layer 4 neurons C_LIO_LIChromosome 21 transcription factors PKNOX1, BACH1, and GABPA drive intellectual disability gene dysregulation C_LIO_LITransplanted human cells model late-stage DS phenotypes, bypassing scarcity of fetal tissue C_LIO_LIASO targeting chromosome 21 transcription factors restores DS-associated molecular signatures C_LI

neuroscience↗
bioRxiv2025

A GCN1-independent activator of the kinase GCN2

Mutations of EIF2AK4, which encodes the eIF2 kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

cell biology↗
bioRxiv2025

Standardization of postmortem human brainstem along the rostrocaudal axis to accommodate for heterogeneity in samples

Human postmortem brain tissues provide an indispensable resource that is crucial for the understanding of neurological conditions, whether related to pathology subtype, burden, distribution or cell-type specificity. Pathology staging protocols provide guidelines for standardized sampling of brain tissues, but cover only a subset of regions affected by pathologies. Thus, to study how various neuropathologies and cell types in highly specialized circuit nodes correlate with functions specifically served by these nodes, additional protocols are necessary. This especially applies to brainstem tissues due to the small dimension of regions of interest and interindividual variability of specimens, whether due to procurement or intrinsic differences. Here we systematically assessed factors contributing to heterogeneity in the length of whole brainstem samples and then presented a standardized approach to reproducibly assign rostrocaudal levels, with standardization relying upon readily identifiable internal landmarks. We validated this approach using postmortem MRI imaging. Standardized brainstem length correlated positively with subject height and negatively with subject age of death. By providing a reference series, reproducible levels can be assigned to individual histological sections or MRI images, i.e. when full brainstem specimens are not available and irrespective of platform, promoting reproducibility.

neuroscience↗
bioRxiv2025

Active surveillance of cats and dogs from households with human COVID-19 cases reveals over one quarter of pets infected with SARS-CoV-2 in 2020-2021 in Texas, United States

Households where people have COVID-19 are high risk environments for companion animals that are susceptible to SARS-CoV-2. We sampled 579 pets from 281 households with one or more laboratory-confirmed person with COVID-19 in central Texas from June 2020 to May 2021. Nineteen out of 396 (4.8%) dogs and 21 out of 157 (13.4%) cats were positive for SARS-CoV-2 by RT-qPCR. Additionally, 95/382 (25%) dogs and 52/146 (36%) cats harbored SARS-CoV-2 neutralizing antibodies. Twenty-six companion animals of ten other species were negative. Overall, 164 (29%) pets were positive for SARS-CoV-2 by molecular and/or serological tests; a total of 110 (39%) out of 281 households had at least one animal with active or past SARS-CoV-2 infection. Cats were more likely to be infected by SARS-CoV-2 and had higher endpoint antibody titers than dogs. Through viral isolation from a subset of respiratory swabs, we documented 6 different lineages in dogs and cats, including the B.1.1 lineage in a cat one month prior to the first known human case in the country. We observed animal and human-pet interaction factors associated with higher risk of infection for dogs and cats, such as days after COVID-19 diagnosis and sharing food. Frequency of clinical signs of disease reported by owners of pets with active infections did not differ from uninfected ones, suggesting that not all reported signs are attributed to SARS-CoV-2 infection. Characterizing animal infections using active SARS-CoV-2 surveillance in pets at risk of infection may aid in One Health pandemic prevention, response, and management.

microbiology↗
bioRxiv2023

Programmed withdrawal of cilia maintenance followed by centriole capping leads to permanent cilia loss during cerebellar granule cell neurogenesis

Primary cilia in brain neurons provide a privileged compartment for binding and responding to extracellular ligands such as sonic hedgehog. Paradoxically, cilia in differentiating cerebellar granule cells are deconstructed during neurogenesis. To identify mechanisms underlying this newly defined cilia deconstruction pathway, we used single cell transcriptomic and immunocytological analyses to compare the transcript and protein signatures of differentiating and progenitor granule cells. We found that differentiating granule cells lacked transcripts for key regulators of pre-mitotic cilia resorption, suggesting cilia disassembly in differentiating cells was distinct from pre-mitotic cilia resorption. Further analysis revealed that during differentiation, transcription of genes required for cilia maintenance decreased. Specifically, protein components of intraflagellar transport complexes, pericentrosomal material and centriolar satellites all decreased as granule cells matured. The changes in transcription and translation correlated with the downregulation of sonic hedgehog signaling at the onset of differentiation. We also found binding of centriolar cap proteins to the mother centrioles as granule cell neurons matured. These data indicate that global, developmentally programmed, diminution of cilium maintenance caused cilia deconstruction in differentiating granule cells. Furthermore, the capping of docked mother centrioles prevents cilia regrowth likely blocking dysregulated sonic hedgehog signaling and tumorigenesis.

cell biology↗
bioRxiv2023

Quantification of circulating tumor DNA using deep learning

Quantification of circulating tumor DNA (ctDNA) levels in blood enables non-invasive surveillance of cancer progression. Fragle is an ultra-fast deep learning-based method for ctDNA quantification directly from cell-free DNA fragment length profiles. We developed Fragle using low-pass whole genome sequence (lpWGS) data from multiple cancer types and healthy control cohorts, demonstrating high accuracy, and improved lower limit of detection in independent cohorts as compared to existing tumor-naive methods. Uniquely, Fragle is also compatible with targeted sequencing data, exhibiting high accuracy across both research and commercial targeted gene panels. We used this method to study longitudinal plasma samples from colorectal cancer patients, identifying strong concordance of ctDNA dynamics and treatment response. Furthermore, prediction of minimal residual disease in resected lung cancer patients demonstrated significant risk stratification beyond a tumor-naive gene panel. Overall, Fragle is a versatile, fast, and accurate method for ctDNA quantification with potential for broad clinical utility.

bioinformatics↗
bioRxiv2023

Single-component multilayered self-assembling protein nanoparticles displaying extracellular domains of matrix protein 2 as a pan-influenza A vaccine

The development of a cross-protective pan-influenza A vaccine remains a significant challenge. In this study, we designed and evaluated single-component self-assembling protein nanoparticles (SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) as vaccine candidates against influenza A viruses. The SApNP-based vaccine strategy was first validated for human M2e (hM2e) and then applied to tandem repeats of M2e from human, avian and swine hosts (M2ex3). Vaccination with M2ex3 displayed on SApNPs demonstrated higher survival rates and lower weight loss compared to the soluble M2ex3 antigen against lethal challenges of H1N1 and H3N2 in mice. M2ex3 I3-01v9a SApNPs formulated with a squalene-based adjuvant were retained in the lymph node follicles over eight weeks and induced long-lived germinal center reactions. Notably, a single low dose of M2ex3 I3-01v9a SApNP formulated with a potent adjuvant, either a Toll-like receptor 9 (TLR9) agonist or a stimulator of interferon genes (STING) agonist, conferred 90% protection against a lethal H1N1 challenge in mice. With the ability to induce robust and durable M2e-specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a SApNP provides a promising pan-influenza A vaccine candidate. ONE-SENTENCE SUMMARYSingle-component self-assembling protein nanoparticles (SApNPs) displaying tandem M2e elicit robust and durable immunity that may protect against influenza A viruses of diverse origins.

microbiology↗
bioRxiv2023

Colorectal cancer detection and treatment with engineered probiotics

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by [~]50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

bioengineering↗
bioRxiv2023

Pathology of natural infection with highly pathogenic avian influenza virus (H5N1) clade 2.3.4.4b in wild terrestrial mammals in the United States in 2022

This article describes the first detections of disease due to natural infection with highly pathogenic avian influenza virus (HPAIv) H5N1 of the Eurasian lineage goose/Guangdong clade 2.3.4.4b in wild terrestrial mammals throughout the United States during 2021-2022. Affected mammalian species include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals primarily exhibited neurological signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

pathology↗
bioRxiv2021

Automated Parallel Pattern Search Optimisation of Microfluidic Geometry for Extracellular Vesicle Liquid Biopsies

Microfluidic liquid biopsies using affinity-based capture of extracellular vesicles (EVs) have demonstrated great potential for providing rapid disease diagnosis and monitoring. However, little effort has been devoted to optimising the geometry of the microfluidic channels for maximum EV capture due to the inherent challenges of physically testing many geometric designs. To address this, we developed an automated parallel pattern search (PPS) optimiser by combining a Python optimiser, COMSOL Multiphysics, and high performance computing. This unique approach was applied to a triangular micropillar array geometry by parameterising repeating unit cells, making several assumptions, and optimising for maximum particle capture efficiency. We successfully optimised the triangular pillar arrays and surprisingly found that simply maximising the total number of pillars and effective surface area did not result in maximum EV capture, as devices with slightly larger pillars and more spacing between pillars allowed contact with slower moving EVs that followed the pillar contours more closely. We then experimentally validated this finding using bioreactor-produced EVs in the best and worst channel designs that were functionalised with an antibody against CD63. Captured EVs were quantified using a fluorescent plate reader, followed by an established elution method and nanoparticle tracking analysis. These results demonstrate the power of automated microfluidic geometry optimisations for EV liquid biopsies and will support further development of this rapidly growing field.

bioengineering↗
bioRxiv2021

One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases - Utah and Wisconsin, 2020

BackgroundApproximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pet cohabitants as a sub-study of an ongoing COVID-19 household transmission investigation. MethodsMammalian pets from households with [≥]1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. Demographic/exposure information, oropharyngeal, nasal, rectal, and fur swabs, feces, and blood were collected from enrolled pets and tested by rRT-PCR and virus neutralization assays. FindingsWe enrolled 37 dogs and 19 cats from 34 of 41 eligible households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dogs fur swabs (2%) tested positive by rRT-PCR at the first animal sampling. Among 47 pets with serological results from 30 households, eight (17%) pets (4 dogs, 4 cats) from 6 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p=0.01). Thirty-three pets with serologic results had frequent daily contact ([≥]1 hour) with the human index patient before the persons COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the human index patient after diagnosis and none (0%) were seropositive; of the 19 (58%) pets with continued contact, 4 (21%) were seropositive. InterpretationsSeropositive pets likely acquired infection from humans, which may occur more frequently than previously recognized. People with COVID-19 should restrict contact with animals. FundingCenters for Disease Control and Prevention, U.S. Department of Agriculture

microbiology↗