Biology subjects

Choi, A.

Publications and source records attributed to Choi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Continuous, high-speed 3D topographic profiling of living tissue monolayers

Tissue monolayers are active living matter whose three-dimensional (3D) architecture and mechanical remodeling regulate tissue function, yet fast and continuous topographic monitoring remains difficult. Here, we present FLuorescence EXclusion microscopy fOr Monolayers (FLEXOM), a microfluidic platform that combines multilayer micropillar arrays with negative-staining optics to convert wide-field images into self-calibrated 3D height maps. Geometry-anchored reference cavities maintain in-frame calibration standards even in fully confluent monolayer fields, enabling continuous 3D profiling from isolated cells to confluent sheets with sub-second temporal resolution ([~]250 ms), sub-micrometer axial precision ([~]0.37 {micro}m), and multi-day biocompatibility and operational stability (>96 h). FLEXOM reveals two distinct anisotropic signatures in tissue monolayers. First, monolayers of different cell types show distinct vertical thickness profiles despite comparable lateral footprints. Only two vertical descriptors, mean height and height variability, distinguished cell types more accurately than nine 2D lateral descriptors, including area, circularity, and convexity (95% vs. 76%). Second, under acute osmotic shock, confluent monolayers exhibit geometrically anisotropic and temporally decoupled vertical-lateral remodeling: height changes dominate the overall volume response and precede lateral remodeling. Surprisingly, cyclic isotonic-hypotonic pulses every 3 min confine remodeling entirely to the vertical axis. Overall, our work provides a high-resolution 3D profiling tool and a framework for axis-resolved analysis of topographic responses in active living matter.

biophysics↗

bioRxiv2026

Bacteriophage treatment of Pseudomonas aeruginosa PA14 infection does not alter the native microbiome of Caenorhabditis elegans

Bacteriophage therapeutics are alternatives to antibiotics for treating multi-drug-resistant (MDR) bacterial infections. Bacteriophages kill host bacteria species with targeted precision, unlike the broad-spectrum nature of antibiotics. While the precision and the natural occurrence of phages in the environment have drawn attention to phages as new "drugs" in the time of accelerated antibiotic resistance, it remains poorly understood how phage treatment impacts the community of organisms composing the microbiome. Before phage therapies can be more widespread in practice, this understanding is required. Caenorhabditis elegans lends itself as an excellent model organism to address this question, as worm populations are raised under sterile conditions and the gut microbiome can be populated experimentally by the worms natural bacterivore diet. Here we have established a C. elegans infection and phage treatment model using fluorescent Pseudomonas aeruginosa PA14 to quantify infection. We isolated and whole genome sequenced PA14-specific lytic phage from wastewater samples near West Point, New York. To characterize the impact of treatment on the microbiome, we populated the C. elegans gut microbiome with 11 strains of the native C. elegans microbiome (CeMbio) and used PacBio sequencing of the full length 16S rRNA gene to characterize the microbiome during PA14 infection in the presence and absence of phage treatment (n = 100 worms per sample, 4 replicates per condition). Taxonomic classification, phylogenetic analysis, and diversity analysis performed using a HiFi 16S bioinformatics pipeline revealed that phage treatment and infection level did not change the overall composition of the C. elegans gut microbiome. ImportanceMultidrug resistant infections are emerging faster than new antibiotic compounds can be synthesized. Bacteriophages are natural viruses of bacteria that seek out and kill only their host bacteria species. Bacteriophage therapeutics are a promising solution for drug resistant infections in patients where no other treatment exists. However, while phage treatment is effective, there is little understanding about what happens within the complex community of the microbiome when a phage treatment is employed. Here we establish a new model utilizing C. elegans to integrate a multidrug resistant infection, phage treatment, and the native microbiome of the C. elegans worm to address how the microbiome changes in response to phage treatment. We find that the microbiome remains unchanged after bacteriophage treatment, highlighting the targeted specificity of phage therapy and suggests fewer side effects for phage therapy than antibiotic treatment for the same infection.

microbiology↗

bioRxiv2026

Development of GS-441524 Derivatives as Potent SARS-CoV-2 Mac1 Inhibitors via a Direct-to-Biology Approach

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

pharmacology and toxicology↗

bioRxiv2026

CD1a-Mediated Presentation of Canonical Microbial Peptides to T Cells

Langerhans cells express the nonpolymorphic antigen-presenting molecule CD1a, positioning them as contributors to host immunity against Mycobacterium leprae in human leprosy. CD1a was originally shown to present non-canonical lipopeptide antigens such as dideoxymycobactin and chemically diverse hydrophobic ligands. Here, we generated CD4 T cell lines from leprosy lesions that recognized M. leprae in a CD1a-restricted manner. Unexpectedly, antigen recognition was protease-sensitive, prompting biochemical purification that identified two microbial protein antigens: LppX, a 25-kDa lipoglycoprotein, and Ag85A, a 30-kDa secreted protein with no known lipid modification. Recombinant proteins activated the corresponding T cell lines in a CD1a-dependent manner. Epitope mapping identified 12-mer peptides that fully reconstituted antigenicity, were conserved between M. leprae and M. tuberculosis, and elicited robust, dose-dependent IFN-{gamma} production and T cell proliferation, establishing that DNA-encoded, ribosomally translated peptides serve as CD1a-restricted cognate antigens. Biochemical analyses showed peptide binding to CD1a, supported by isoelectric focusing and surface plasmon resonance (KD [~]75 M for Ag85A). CD1a-peptide tetramers specifically stained cognate T cells, soluble CD1a was sufficient to present peptide antigen, and transfer of the LppX-specific TCR into naive T cells restored antigen responsiveness. Using CD1a-peptide tetramers, we identified antigen-specific T cells enriched in patients undergoing reversal reactions compared with patients with lepromatous leprosy and healthy donors. The CD1a-restricted T cell lines secreted IFN-{gamma} and IL-26, cytokines with established antimicrobial activity. Together, these findings demonstrate that CD1a can present canonical microbial peptides as part of a cell-mediated immune response in leprosy, extending the known spectrum of CD1a ligands. Because CD1a is nonpolymorphic and presents antigens to antimicrobial T cells, CD1a-peptide complexes may provide a broadly applicable platform for studying, detecting, and potentially targeting mycobacterial immunity.

immunology↗

bioRxiv2026

A retrospective public external benchmark of healthy-to-stroke lower-limb EEG transport identifies constraints from source construction, adaptation burden, and confound sensitivity

BackgroundLower-limb EEG is a rehabilitation-facing control signal for stroke neurorehabilitation and future non-invasive brain-spine interfaces, but a public external benchmark that jointly audits source construction, minimal adaptation burden, and confound sensitivity is lacking. We therefore tested whether lower-limb effort-versus-rest decoders trained on healthy public EEG transport to a stroke target domain. MethodsWe conducted a retrospective public-data external benchmark using three public EEG datasets harmonised to a common lower-limb effort-versus-rest target. Classical and deep models were compared under zero-shot transport, 10-shot temperature calibration, and 10-shot fine-tuning. For few-shot analyses, each target participant contributed a trial-disjoint subject-internal support set of 10 labelled trials per class and a held-out remainder test set. Prespecified analyses audited source construction, support-resampling sensitivity, and montage controls. Uncertainty was summarised with participant-level bootstrap confidence intervals. ResultsWithin this benchmark, healthy-to-stroke zero-shot transport was weak. The best zero-shot result was classical rather than deep, with CSP+LDA reaching area under the receiver operating characteristic curve (AUROC) 0.603, whereas EEGNet remained near chance (AUROC 0.527). Ten-shot calibration improved operating behaviour more than discrimination: for CSP+LDA, expected calibration error fell from 0.267 to 0.035 and specificity increased from 0.180 to 0.485, whereas AUROC remained essentially unchanged (0.603 to 0.604). Ten-shot fine-tuning produced only modest gains; the best overall AUROC was 0.605 for pooled dataset-balanced CSP+LDA, numerically tied with pooled raw CSP+LDA (0.605). MILimbEEG-only source training was consistently weak, exploratory deep domain-generalisation variants did not rescue transport, and frontal and temporal montage controls remained relatively competitive. ConclusionsWithin this public benchmark, source construction and minimal adaptation burden mattered more than model novelty, and retrospective montage controls limited motor-specific interpretation. The results support harmonised prospective validation of lower-limb EEG transport over further retrospective model iteration.

neuroscience↗

bioRxiv2026

Lung endothelial niche signaling governs self-renewal and fate transitions of human alveolar stem cells

Chronic lung diseases such as pulmonary fibrosis are characterized by the irreversible loss of alveolar type 1 (AT1) cells, yet the mechanisms governing human alveolar stem cell self-renewal and differentiation remain poorly defined. Here, we identify a lung endothelial niche that sustains the self-renewal of human alveolar type 2 (AT2) stem cells through MAPK signaling, enabling robust long-term expansion while preserving stem cell fate. Although YAP activation initiates AT1 transcriptional programs, it is insufficient to complete lineage maturation. We show that MAPK inhibition together with LATS inhibition promotes nuclear translocation of YAP, enhancing AT1 differentiation. Expanded human AT2 stem cells engraft in fibrotic lungs and contribute to alveolar regeneration while undergoing directed differentiation within diseased human lung tissue. Together, our findings define a niche-controlled signaling mechanism governing human alveolar stem cell fate and advance our understanding of alveolar regeneration.

cell biology↗

bioRxiv2026

Fail closed trust gated synthetic augmentation governs tail risk under subject shift in EEG

Synthetic augmentation can silently harm subject-disjoint EEG generalization. We propose trustgated augmentation (TGA), a control layer that scores synthetic windows with a teacher trained on real data for label consistency and confidence; only samples above a confidence quantile q are eligible. A fail-closed selector injects synthetic data only if validation AUROC exceeds real-only by a margin, otherwise reverting to real-only. In PainMunich chronic-pain EEG (n = 189) at 5% subject scarcity, ungated augmentation harmed 56% of paired runs ({Delta}AUROC< -0.01), whereas TGA at q = 0.99 reduced harm to 24% with comparable mean AUROC. In BCI IV-2a motor imagery (n = 9) at 25% scarcity, strict gating improved AUROC (0.679 vs 0.627) and reduced harm (0.16 vs 0.44). A covariance-manifold audit showed synthetic windows were strongly off-manifold (mean distance ratio 2.39 x 104), motivating explicit governance.

bioinformatics↗

bioRxiv2025

Electronic Cigarette Aerosol Exposure Induces Airway Remodeling in 3D Human Tracheobronchial Epithelial Tissues: From Goblet Cell Hyperplasia to Squamous Metaplasia

Electronic cigarette (EC) aerosols are increasingly recognized for their disruptive effects on airway physiology, yet the temporal progression of epithelial remodeling remains poorly characterized. While squamous metaplasia, mucin hypersecretion, and ciliary dysfunction are hallmark features of smoking-related airway disease, analogous data for EC exposure are limited. To address this gap, we employed a 3D human tracheobronchial epithelial tissue (hTET) model derived from airway basal stem cells and exposed it daily to NJOY ACE "Classic Tobacco" aerosols (1 or 5 puffs) or clean air for 1, 2, or 3 weeks. Remodeling was assessed via immunolabeling and Western blot analysis. MUC5AC expression revealed a transient hypersecretory response, peaking at week 2 and declining by week 3. Multiparametric analysis demonstrated progressive squamous remodeling: early ciliary loss and involucrin upregulation emerged by week 1, with severity increasing in a puff-dependent manner. By week 2, tissues exhibited marked ciliary depletion, altered epithelial thickness, horizontal nuclear reorientation, and elevated involucrin. At week 3, both exposure groups developed a squamous metaplasia phenotype, characterized by involucrin-positive squamous cells replacing ciliated cells. Quantitative analysis confirmed sustained involucrin elevation, with the 1-puff group reaching levels comparable to the 5-puff group by week 3. These findings delineate a sequential pathological trajectory initiated by EC aerosols, progressing from transient goblet cell activation to squamous metaplasia. This human-relevant model underscores the potential for repeated low-level EC exposure to induce airway epithelial remodeling and pathology.

pharmacology and toxicology↗

bioRxiv2025

Inflammatory reprogramming of human brain endothelial cells compromises blood-brain barrier integrity in Alzheimer's disease

Blood-brain barrier (BBB) dysfunction is an early feature of Alzheimers disease (AD), yet the endothelial gene-regulatory programs involved remain incompletely understood. We integrate postmortem human single-nucleus transcriptomics with iPSC-based BBB models to define a conserved, inflammation-driven pathway that compromises barrier integrity. We identify an NF-{kappa}B-associated endothelial gene module endoM2 that is elevated in AD, inversely correlated with cognition, and enriched for inflammation and endothelial-to-mesenchymal transition signatures. Cytokine stimulation of iPSC-derived brain endothelial cells induces morphological remodeling, lipid accumulation, junctional disruption, and transcriptomic shifts that mirror endoM2. A targeted drug screen identifies the NF-{kappa}B inhibitor BAY11-7082 as protective against cytokine-induced changes. In our perfusable iPSC-derived BBB-Chip that recapitulates human BBB signatures, single-cell profiling reveals inflammatory endothelial state-specific programs reflecting those in AD brains and demonstrates that BAY11-7082 suppresses cytokine-triggered dysfunction and reverses inflammation-associated gene activation. Together, these findings position cerebrovascular inflammation as a therapeutic target to preserve BBB integrity in AD.

neuroscience↗

bioRxiv2025

Vascular-Perfusable Human 3D Brain-on-Chip

Development and delivery of treatments for neurological diseases are limited by the tight and selective human blood-brain barrier (BBB). Although animal models have been important research and preclinical tools, the rodent BBB exhibits species differences and fails to capture the complexity of human genetics. Microphysiological systems incorporating human-derived cells hold great potential for modeling disease and therapeutic development, with advantages in screening throughput, real-time monitoring, and tunable genetic backgrounds when combined with induced pluripotent stem cell (iPSC) technology. Existing 3D BBB-on-chip systems have incorporated iPSC-derived endothelial cells but not the other major brain cell types from iPSCs, each of which contributes to brain physiology and disease. Here we developed a 3D Brain-Chip system incorporating endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia from iPSCs. To enable this multicellular 3D co-culture in-chip, we designed a GelChip microfluidic platform using a 3D printing-based approach and dextran-based engineered hydrogel. Leveraging this platform, we co-cultured and characterized iPSC-derived brain-on-chips and modeled the brain microvasculature of APOE4, the strongest known genetic risk factor for sporadic Alzheimers disease. These 3D brain-on-chips provide a versatile system to assess BBB vascular morphology and function, investigate downstream neurological effects in disease, and screen therapeutics to optimize delivery to the brain. Significance StatementThe blood-brain barrier (BBB) is both a contributing factor to neurological disease and a major obstacle to its treatment, yet human-relevant models remain limited. Most existing brain-on-chip systems incorporate only subsets of BBB cell types and cannot capture the full cellular complexity of the human neurovascular unit. Here, we establish a vascular-perfusable 3D Brain-Chip using human induced pluripotent stem cell-derived brain cells including endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia. This system enables systematic analysis of human genetic risk factors, such as APOE4 in Alzheimers disease, and provides a powerful platform to investigate BBB function and dysfunction and accelerate the development of more effective neurological therapies.

neuroscience↗

bioRxiv2025

CloneCoordinate: Open-source software for collaborative DNA construction

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.

synthetic biology↗

bioRxiv2025

COVID-19 induces persistent transcriptional changes in adipose tissue that are not associated with Long COVID

Long COVID is a heterogeneous condition characterized by a wide range of symptoms that persist for 90 days or more following SARS-CoV-2 infection. Now more than five years out from the onset of the SARS-CoV-2 pandemic, the mechanisms driving Long COVID are just beginning to be elucidated. Adipose tissue has been proposed as a potential reservoir for viral persistence and tissue dysfunction contributing to symptomology seen in Long COVID. To test this hypothesis, we analyzed subcutaneous adipose tissue (SAT) from two cohorts: participants with subacute COVID-19 (28-89 days post-infection) compared to pre-pandemic controls, and participants with Long COVID compared to those with those classified as "indeterminate" based on the RECOVER-Adult Long COVID Research Index (12-47 months post-infection). We found no evidence of persistent SARS-CoV-2 RNA in adipose tissue in any participant. SAT from participants with subacute COVID-19 displayed significant transcriptional remodeling, including depleted immune activation pathways and upregulated Hox genes and integrin interactions, suggesting resident immune cell exhaustion and perturbations in tissue function. However, no consistent changes in gene expression were observed between Long COVID samples and samples from indeterminant participants. Thus, SAT may contribute to inflammatory dysregulation following COVID-19, but does not appear to play a clear role in Long COVID pathophysiology. Further research is needed to clarify the role of adipose tissue in COVID-19 recovery.

immunology↗

bioRxiv2025

Deletion of a KSF Motif Attenuates NSP1 Host Cell Translation Shutoff and Impairs SARS-CoV-2 Virulence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion ({Delta}KSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo, similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same {Delta}KSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. IMPORTANCEUnderstanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the hosts immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.

microbiology↗

bioRxiv2024

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation enhanced host lipid droplet consumption by NSP6 without impacting its known ER-zippering function or double-membrane vesicle morphology. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6s key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications. Author SummaryAs SARS-CoV-2 continues to spread and adapt in humans, viral variants with enhanced spread and immune evasion have emerged throughout the COVID-19 pandemic. While most of the mutations occur in the Spike protein and have been extensively studied, non-Spike mutations have been accumulating and are not as well understood. Here, we constructed Spike-defective genomes of recent Omicron variants and systematically compared their RNA replication capabilities independently from Spike. We also performed single-round infection assays with various Omicron variant Spike proteins to quantify viral entry capabilities. Interestingly, viral entry and RNA replication were negatively correlated, suggesting that as variants evolved reduced entry functions under growing immune pressure on Spike, RNA replication increased as a compensatory mechanism. We focused on a viral protein NSP6 that acquired mutations that significantly enhanced RNA replication. We validated that a frequently accessed L260F mutation in NSP6 enhanced viral infection in cells and increased pathogenesis in mice. While the mutation did not alter NSP6s role in maintaining a membranous network of viral replication factories, the mutation enhanced viral hijacking of the host lipid droplet machinery. Collectively, we highlight the important role of non-Spike mutations in the evolutionary trajectories of SARS-CoV-2 variants with possible monitoring and therapeutic implications.

microbiology↗

bioRxiv2024

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused [~]50% slower, while diffusion of positively charged proteins was reduced by [~]80-90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, negatively charged proteins of different sizes showed similar diffusivity suppression in extract, which are typically prepared under conditions that inhibit actin polymerization. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks. Significance StatementThe complex intracellular molecular environment is notably challenging to elucidate and recapitulate. Xenopus egg extracts provide a native yet manipulatable cytoplasm model. Through single-molecule microscopy, here we decipher the cytoplasmic environment and molecular interactions by examining the diffusion patterns of diverse proteins in Xenopus egg extracts with strategic manipulations. These experiments reveal an overwhelmingly negatively charged macromolecular environment with crosslinked meshworks, offering new insight into the inner workings of the cell.

biophysics↗

bioRxiv2024

Molecular basis of pathogenicity of the recently emerged FCoV-23 coronavirus

The ability of coronaviruses to recombine and cross species barriers affects human and animal health globally and is a pandemic threat. FCoV-23 is a recently emerged, highly pathogenic recombinant coronavirus responsible for a widespread outbreak of feline infectious peritonitis (FIP) likely linked to in-host viral evolution. Here, we report cryoEM structures of two FCoV-23 spike (S) isoforms explaining that the in-host loss of domain 0 observed in clinical samples enhances entry into cells and fusogenicity by facilitating protease access, leading to biotype switching and lethality. We show that FCoV-23 can use several aminopeptidase N (APN) orthologs as receptors and reveal the molecular determinants of receptor species tropism, including a glycan modulating human receptor utilization. We define antigenic relationships among alphacoronaviruses infecting humans and other mammalian species and identify a cross-reactive alphacoronavirus monoclonal antibody inhibiting FCoV-23 pseudovirus entry, paving the way for vaccine and therapeutic development targeting this highly pathogenic virus.

biochemistry↗

bioRxiv2024

Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion

As with most intermediate filament systems, the hierarchical self-assembly of vimentin into nonpolar filaments requires no nucleators or energy input. Utilizing a set of live-cell, single-molecule, and super-resolution microscopy tools, here we show that in mammalian cells, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing differently charged constructs, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges (-18 per vimentin protein) along the filament are less screened or otherwise intensified, and stabilized when the charges are better screened or otherwise reduced. Generalizing this model to other intermediate filaments, we further show that whereas the negatively charged GFAP cytoskeleton is similarly subject to fast disassembly under hypotonic stress, the cytokeratin, as a copolymer of negatively and positively charged subunits, does not exhibit this behavior. Thus, in cells containing both vimentin and keratin cytoskeletons, hypotonic stress disassembles the former but not the latter. Together, our results both provide new handles for modulating cell behavior and call for new attention to the effects of net charges in intracellular protein interactions.

biophysics↗

bioRxiv2024

SARS-CoV-2 serosurvey of healthy, privately owned cats presenting to a New York City animal hospital in the early phase of the COVID-19 pandemic (2020-2021)

SARS-CoV-2, the cause of the ongoing COVID-19 pandemic, not only infects humans but is also known to infect various species, including domestic and wild animals. While many species have been identified as susceptible to SARS-CoV-2, there are limited studies on the prevalence of SARS-CoV-2 in animals. Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the U.S. in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health "lock-down". Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and two of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1/1024 and 1/4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV-2 in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our study provide a new context for SARS-CoV-2 transmission events across species. SignificanceSARS-CoV-2 has a broad animal tropism and can infect a wide range of animal species, leading to an expansion of the viral reservoir. Expansion of this viral reservoir may result in the accumulation of mutations within these species, potentially giving rise to new viral variants and facilitating reverse zoonotic transmission. Domestic cats are particularly noteworthy in this regard due to their close contact with humans. Currently, there are very limited studies on the prevalence of SARS-CoV-2 infection in domestic cats during the early stages of the pandemic, especially in the United States. This retrospective study addresses the gap by investigating seroprevalence of SARS-CoV-2 in cats in New York City, the epicenter of the COVID-19 pandemic in the United States during the early pandemic. Our work underscores the importance of adopting a One Health approach to pandemic prevention and conducting routine surveillance across different animal species

microbiology↗