Biology subjects

Guo, R.

Publications and source records attributed to Guo, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Structure and Mechanism of a Two-component Lanthipeptide Toxin

The enterococcal cytolysin is a two-component lanthipeptide natural product that co-operatively kills mammalian cells and bacteria. It is a virulence factor in typically commensal strains of gut-dwelling Enterococcus faecalis and causes a fatal form of alcoholic hepatitis. Despite its clinical significance, little is known about the mechanism of action. We present here the molecular details of cytolysins remarkable bioactivity. A high-resolution cryo-electron microscopy structure revealed highly ordered tubular assemblies comprising the two subunits. We further demonstrate these structures disrupt the membranes of eukaryotic and bacterial cells. These results provide the first high-resolution structure of two distinct lanthipeptides interacting with one another and offer an explanation for the unique bioactivity of the cytolysin toxin.

biochemistry↗

bioRxiv2026

Pan-cancer virtual spatial transcriptomics from routine histology with Phoenix

Spatial transcriptomics links gene expression to tissue architecture, providing a mechanistic view of cellular organization. Yet existing datasets cover few donors and miss the complexity of human disease. Experimental costs remain prohibitive, and large-scale profiling is impractically slow for population-level studies. Accurate computational methods are urgently needed. Predicting gene expression from standard histology, however, remains an open problem, as current approaches transfer poorly to unseen cohorts and diseases. Here, we present Phoenix, a (latent) flow matching generative model that infers pan-cancer spatially resolved single-cell gene expression with high accuracy. Phoenix analyzes treatment response in silico: Applied to 763 head and neck cancer patients, it identified three new spatial biomarkers that we validated across two cancers (breast cancer, n = 84; ovarian cancer, n = 157) and treatment regimens (platinum, trastuzumab). Phoenix generalizes beyond carcinomas: In a large sarcoma cohort (802 tissue microarray cores), it accurately predicted cell-type-specific signatures in held-out samples and captured chemotherapy-induced immune remodeling. Phoenix also extends across species: In a mouse model, it accurately predicted the expression of pancreatic cancer lineage markers and the mutant mKras^G12D allele in silico. In total, we evaluated Phoenix on over 10,000 patients. Our results establish virtual spatial transcriptomics as a scalable framework for studying tissue organization, therapeutic response, and disease mechanisms.

bioinformatics↗

bioRxiv2026

Domain classification of archaeal proteomes reveals conserved fold repertoire

Archaea represent one of the three domains of cellular life and yet account for fewer than 1% of experimentally determined protein structures, leaving the extent of their structural novelty unknown. Here we present a systematic domain-level classification of 124,075 proteins from 65 archaeal classes spanning 21 phyla and all major lineages, using both AFDB and newly predicted AlphaFold3 structures classified against the Evolutionary Classification of protein Domains (ECOD). We assigned 204,758 domains, of which 76.8% received high-confidence classifications, spanning 987 ECOD X-groups; 40% of known structural diversity within a single domain of life. Clustering by Foldseek recovered structural relationships for 63% of domains that are singletons by sequence comparison. To characterize the 21% of proteins lacking high-confidence classification, we applied successive filters for structure prediction confidence, protein length, and structural cluster context, reducing 8,452 domain-free proteins to a small number of well-folded structural orphans (less than 0.1% of the dataset). The unclassified fraction is dominated by sub-threshold matches to known folds (14% of all proteins) and low-confidence structure predictions (5%), not by novel structures. These results demonstrate that the protein fold repertoire at the single-domain level is broadly conserved across the deepest phylogenetic distances in cellular life, and that the gap between archaeal and well-characterized proteomes reflects classification sensitivity for divergent sequences rather than unexplored structural diversity.

bioinformatics↗

bioRxiv2026

Epstein-Barr Virus Latent Membrane Protein 1 Suppresses Ferroptosis via Pentose Phosphate Pathway and Glutathione Metabolism

Epstein-Barr virus (EBV) is associated with 200,000 cancers per year, including Burkitt lymphoma and post-transplant lymphomas. We previously reported that EBV latency oncogene programs dynamically remodel infected B cell metabolism and sensitivity to induction of ferroptosis, a programmed cell death pathway driven by lipid reactive oxygen species. However, much has remained unknown about how EBV remodels key redox defense pathways in support of infected B cell proliferation. Here, we identify EBV latent membrane protein 1 (LMP1), a key viral oncogene necessary for B cell immortalization and which mimics aspects of CD40 signaling, drives resistance to ferroptosis induction by erastin, a small molecule that blocks cystine uptake. LMP1 expression was sufficient to protect Burkitt cells from erastin ferroptosis induction. Mechanistically, signaling from the LMP1 TES2/CTAR2 region drove this phenotype, which was not shared by CD40 signaling, revealing that LMP1 evolved independent redox defense roles. Metabolomic analysis highlighted key LMP1 and TES2 signaling roles in support of antioxidant cysteine and glutathione levels. TES2 signaling supported cystine uptake, glutathione and NADPH pools in newly infected peripheral blood B cells. We identified PFKFB4, a host enzyme that shunts glucose into the pentose phosphate pathway to support NADPH production, as a major TES2 metabolic target. PFKFB4 knockdown increased EBV-transformed lymphoblastoid cell line lipid ROS levels, decreased glutathione and strongly sensitized them to ferroptosis induction by erastin treatment. PFKFB4 was also necessary for LMP1-mediated Burkitt B cell ferroptosis resistance. Collectively, these results identify PFKFB4 as a key host cell EBV metabolism remodeling target critical for infected B cell redox defense.

microbiology↗

bioRxiv2026

Sub-second Extracellular Impedance Measurement of Epithelial Cell Monolayers using Step Excitations and Time-domain Analysis

Extracellular electrochemical impedance spectroscopy (EIS) is emerging as a powerful technique in in vitro epithelial research, offering quantitative insights into barrier integrity, morphology, and apical-basolateral polarity noninvasively through metrics such as transepithelial electrical resistance (TER/TEER), transepithelial capacitance (TEC), and membrane ratio (), respectively. However, due to the broad range of frequencies probed, EIS typically requires tens of seconds per measurement, limiting its ability to capture more rapid biological phenomena. We present Time-domain Epithelial Impedance Measurement (TEIM), a method for sub-second extracellular impedance measurements of epithelial cell monolayers based on step (Heaviside function) current excitation and time-domain analysis of the voltage transients, without the need for Fourier transforms. We experimentally demonstrate TEIM measuring TER, TEC, , and model-derived impedance spectrum at [~]0.3 s sampling rate, which represents a 100- fold improvement in time resolution compared with traditional EIS. The accuracy and precision of TEIM were benchmarked against EIS on both electrical circuits and epithelial cell monolayers of immortalized Human Bronchial Epithelial (16HBE) and Human Colorectal Adenocarcinoma (Caco-2) (n = 3 for each), and average percent errors for TER, TEC, and ranged from 0.17-3.55%, 1.13-8.96%, and 0.59-26.35%, respectively. Application of TEIM to monitor Caco-2 responses to saponin, a quick-acting pore-forming detergent, revealed smoothly gated double-exponential transient TER and TEC dynamics that were too rapid to be adequately captured previously using EIS. Overall, TEIM enables electrophysiology studies of rapid changes in epithelial cell culture models and possibly more complex in vitro models, holding promise for future applications in areas such as disease modeling, therapeutic development, and beyond.

bioengineering↗

bioRxiv2026

A human-in-the-loop explanation framework for morphologically transparent AI predictions from whole-slide images

Deep learning models enable the prediction of clinical endpoints from whole-slide images (WSIs), but many such models function as "black boxes", lacking transparency about whether and which histomorphological patterns drive their predictions, hindering interpretability and clinical adoption. Here we propose a human-in-the-loop explanation framework, MorphoXAI, which provides both local and global interpretability for deep learning models by incorporating human-expert interpretations. At the global level, it reveals the histomorphological patterns on which the model consistently relies to distinguish between classes of WSIs, as well as the patterns associated with confusion between classes. At the local level, it indicates which of these patterns are used in the prediction of an individual WSI and which regions within the slide correspond to such patterns. We validated our method on a deep learning model trained for ovarian tumor histologic subtype prediction. The results show that our framework generates explanations that accurately reflect the histomorphology underlying the models predictions at both global and local levels. For interpretability and clinical utility in diagnostic contexts, human evaluation results showed that our explanations were easy to interpret, rich in diagnostic features, and directly helpful for diagnostic decision-making, thereby enhancing pathologist-AI collaboration. Our work highlights that unifying global and local explanations and grounding them in expert-interpreted morphology enhances the interpretability and verifiability of deep learning models, thereby facilitating the transparent deployment of such models in clinical practice.

pathology↗

bioRxiv2026

Establishing the molecular basis for MDA5 mutation-linked autoimmunity

Melanoma differentiation-associated protein 5 (MDA5), a member of the RIG-I-like receptor family, is a cytoplasmic sensor essential for innate antiviral immunity. MDA5 distinguishes viral RNA from host RNA in part through its ATP hydrolysis activity, which promotes filament turnover on shorter endogenous dsRNAs. Here, we show that the gain-of-function T331I disease-linked mutation within the ATP binding pocket disrupts this balance, resulting in constitutive interferon signaling. Through a combination of cryo-electron microscopy (cryoEM), biochemical assays, and cellular analyses, we reveal the extensive network of interactions that precisely position ATP for catalysis in the wild-type MDA5 ATP binding pocket, and also demonstrate that the T331I mutation impairs ATPase activity, thereby stabilizing MDA5-dsRNA complexes and leading to aberrant immune activation. These findings elucidate how MDA5 ATPase activity regulates antiviral specificity and prevents autoimmunity by controlling filament stability and downstream signaling, offering a mechanistic molecular explanation for disease pathogenesis.

biochemistry↗

bioRxiv2026

Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified Intra-tumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based TME classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13 CD4 T cells co-localize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudo-temporal maturation continuum: aggregated, activated, and post-activated. Intra-niche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12 macrophages, evolved dynamically with maturation. Single-nucleus RNA-sequencing combined with Tangram-based spatial mapping revealed CXCL12 macrophages and iCAFs forming a peripheral band in aggregated TLSs, while ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy. TeaserAn atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.

immunology↗

bioRxiv2025

SF3A2 deacetylation at lysin 10 mitigates myocardial pathological remodeling by reprogramming cardiomyocyte metabolism

BackgroundDysregulation of splicing factor-mediated pre-mRNA alternative splicing (AS) underlies the progression of complex diseases, but the specific involvement of AS in myocardial pathological remodeling remains unclear. This study aimed to elucidate the role of splicing factor 3A, subunit 2 (SF3A2) in the development of myocardial pathological remodeling by regulating AS and its effect on metabolic reprogramming. MethodsWe evaluated the function and mechanism of SF3A2 in myocardial pathological remodeling using murine models induced by myocardial infarction (MI) and isoproterenol. The gain- and loss-of-function models (AAV9-cTNT delivery-mediated knock-in and lentivirus-mediated knockdown) combined with acetylomics, metabolomics, lipidomics, 13C-glucose tracing and immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (IP-MS) analyses were used to demonstrate the mechanism of SIRT7-mediated SF3A2 deacetylation at lysin 10 (K10). Unbiased RNA sequencing and AS analysis were conducted to identify the downstream effector of SF3A2. ResultsSF3A2 acetylation at lysine 10 was upregulated in cardiomyocytes in response to myocardial pathological remodeling. Functionally, the knock-in of a deacetylation-mimetic SF3A2 (K10R) mutant attenuated myocardial hypertrophy, fibrosis, and improved heart function. These phenotypes were accompanied by a redirection of glucose flux from anaerobic glycolysis to the TCA cycle, along with a promotion of fatty acid {beta}-oxidation by inhibiting CD36 translocation and decreasing ACLY activity. Mechanistically, SF3A2 mitigated metabolic reprogramming and mitochondrial dysfunction by regulating Nnt AS, a process involving the SF3A2-interacting protein Ddx1. IP-MS further identified SIRT7 as the deacetylase for SF3A2, which was found to be activated by ginsenoside Rg1 in a high-throughput screen, leading to improved heart function. ConclusionsOverall, our findings establish the SIRT7-SF3A2-Nnt axis as a critical regulator in metabolic reprogramming to delay pathological myocardial remodeling. Promoting SF3A2 deacetylation, mediated by AAV9 delivery or pharmacological activation of SIRT7, improved heart function, suggesting its potential as a therapeutic target for pathological myocardial remodeling.

molecular biology↗

bioRxiv2025

Dual-Channel Event Microscopy for Ultrafast Biological Imaging

Many fundamental biological processes--spanning immune-tumor interactions, neuronal signaling, and microvascular flow--exhibit fast, multiscale dynamics among diverse cell types within three-dimensional tissue environments. Capturing such activity requires imaging systems that simultaneously achieve high temporal resolution, multicolor capability, and volumetric coverage over large fields of view (FOVs). However, existing modalities remain limited by trade-offs among imaging speed, spectral capacity, depth of field (DOF), and spatial resolution. Here, we present Dual-Channel Event Microscopy (DEM), which integrates digital micromirror device (DMD)-based pulsed illumination, extended-DOF optics, and event-based sensing for ultrafast, dual-channel volumetric imaging across a 2.3 x 1.3 mm2 FOV with an effective 200 {micro}m DOF. Using dual-color fluorescent phantoms and microsphere flow assays, DEM achieves accurate spectral separation and reconstruction of rapid motion at kilohertz frame rates. In vivo, DEM enables simultaneous visualization of neutrophils and premalignant tumors in freely swimming zebrafish. In immobilized specimens, it provides robust, sensor-level optical sectioning near the heart, suppressing diffuse background to reveal fine vascular networks and active blood circulation into and out of the cardiac chambers. DEM further enables quantitative mapping of blood-flow dynamics in the zebrafish tail, resolving arterial-venous differences and capturing heartbeat-driven oscillations that reflect cardiac pumping with high temporal fidelity. By uniting ultrafast acquisition, dual-channel capability, volumetric coverage, and intrinsic optical sectioning within a single event-driven architecture, DEM offers a powerful platform for visualizing rapid multicellular interactions and physiological dynamics in living systems.

bioengineering↗

bioRxiv2025

Epstein-Barr Virus Encoded lncRNAs Control the Viral Lytic Switch

Epstein-Barr virus (EBV) uses a biphasic lifecycle, switching between latent and lytic phases to persistently infect most adults. Latency is observed in most tumor cells of the 200,000 EBV-associated cancers/year. EBV reactivation is increasingly implicated in autoimmune diseases, including multiple sclerosis. However, mechanisms that regulate EBV reactivation have remained incompletely understood. Here, we leveraged multi-omic approaches to reveal the existence of pro-latency and a pro-lytic viral long noncoding RNAs (lncRNAs) that counter-regulate the lytic switch. Known reactivation triggers rapidly induced expression of the pro-lytic lncRNA, which encodes an RNA G-quadruplex that mediated its interaction with CTCF. The pro-lytic lncRNA occupies viral origin of lytic replication enhancers and promotes their looping to the immediate early lytic promoter to trigger reactivation. The pro-latency lncRNA duplexes with the pro-lytic RNA to impede its interactions with CTCF. These studies lay a foundation for therapeutic approaches to manipulate the EBV lytic switch.

microbiology↗

bioRxiv2025

Dimethylindanoylindole isomers revealed different pharmacological profiles at CB1 and CB2 cannabinoid receptors

Synthetic cannabinoid receptor agonists (SCRAs) represent one of the most rapidly growing classes of novel psychoactive substances (NPS) and exhibit higher efficacy and potency at CB1 and CB2 cannabinoid receptors (CB1 and CB2) compared to {Delta}9-tetrahydrocannabinol, contributing to distinct adverse effects absent in cannabis use. The indole-based SCRAs GBD-002 and GBD-003, incorporating 2,2-dimethylindane, differ by a single substituent position yet display markedly different receptor binding profiles. We evaluated both the GBD-002/GBD-003 pair and an analogous structural isomer pair using bioluminescence resonance energy transfer (BRET) assays to assess key CB1 and CB2 transducer pathways. Site-directed mutagenesis characterized contributions of non-conserved residues between CB1 and CB2 and aromatic residues on CB1 transmembrane helix 2 (TM2). This study provided insights into the molecular determinants of cannabinoid receptor selectivity and efficacy.

pharmacology and toxicology↗

bioRxiv2025

Construction of a novel 3D urinary bladder mucosa model and its application in toxicity assessment of arsenicals

The urinary bladder is a major target organ for environmental toxicants, including arsenic. The objects of this study were two-fold. First, we constructed a novel 3D urinary bladder mucosa model that incorporated an overlying epithelium and a supporting subepithelial layer, referred to as the 3D-UBMM. Primary human bladder urothelial and fibroblast cells were immortalized by introducing the human CDK4R24C and TERT genes. The subsequent construction of the 3D-UBMM involved incorporating the immortalized fibroblast cells into a collagen raft, while the immortalized urothelial cells were cultured at the air-liquid interface of the raft. This 3D-UBMM closely resembles the human urinary bladder epithelium in terms of morphology and marker protein expression, including uroplakin 1b, P63, and cytokeratin 5. Second, using the 3D-UBMM we investigated the cytotoxicity of sodium arsenite (iAsIII) and dimethylarsenic acid (DMAV). Exposure to iAsIII and DMAV resulted in increased urothelial cell necrosis and increased {gamma}-H2AX-positive cells along with a reduction of P63-positive cells, and each of these effects was induced in a dose-response manner. These findings affirm that this novel 3D-UBMM closely resembles the human urinary bladder epithelial layer, offering a practical in vitro model for the evaluation of the toxicity of arsenic and other bladder carcinogens and the role of cancer-related genes in bladder carcinogenicity. In addition, by identifying mechanisms of carcinogenesis this model will aid in hazard identification and risk assessment of potential bladder carcinogens.

pharmacology and toxicology↗

bioRxiv2025

Developing a Novel In Vitro Toxicity Assay for Predicting Inhalation Toxicity in Rats

The development of alternative in vitro methods for assessing acute inhalation toxicity is a critical step toward reducing animal testing and aligns with the principles of the 3Rs (replacement, reduction, and refinement). In this study, we developed and optimized a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. To improve assay efficiency and robustness, we introduced two key modifications: the incubation time was reduced to 15 minutes to enable rapid and high-throughput screening, and for chemicals reactive with polystyrene 6-well glass plates were used to prevent chemical-induced degradation and ensure assay consistency. LC50 values were determined for 49 chemicals and compared with reported LC50 values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC50 values and in vivo LC50 values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC50 values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC50 values showed significant negative correlations with molecular weight and octanol-water partition coefficients, indicating that chemicals with higher values tended to be less cytotoxic in vitro. Importantly, the A549-NRU assay demonstrated stronger correlation with in vivo LC50 values than the conventional NRU assay using mouse 3T3 fibroblast cells. These findings support the use of the A549-NRU assay to estimate starting doses for in vivo studies, and potentially as an in vitro alternative for predicting acute inhalation toxicity. Impact statementThe optimized A549-NRU assay demonstrates predictive potential for inhalation toxicity while reducing reliance on animal testing. This model serves as a human-relevant alternative for estimating starting doses for in vivo inhalation toxicity studies.

pharmacology and toxicology↗

bioRxiv2025

GAME: Genomic API for Model Evaluation

The rapid expansion of genomics datasets and the application of machine learning has produced sequence-to-activity genomics models with ever-expanding capabilities. However, benchmarking these models on practical applications has been challenging because individual projects evaluate their models in ad hoc ways, and there is substantial heterogeneity of both model architectures and benchmarking tasks. To address this challenge, we have created GAME, a system for large-scale, community-led standardized model benchmarking on user-defined evaluation tasks. We borrow concepts from the Application Programming Interface (API) paradigm to allow for seamless communication between pre-trained models and benchmarking tasks, ensuring consistent evaluation protocols. Because all models and benchmarks are inherently compatible in this framework, the continual addition of new models and new benchmarks is easy. We also developed a Matcher module powered by a large language model (LLM) to automate ambiguous task alignment between benchmarks and models. Containerization of these modules enhances reproducibility and facilitates the deployment of models and benchmarks across computing platforms. By focusing on predicting underlying biochemical phenomena (e.g. gene expression, open chromatin, DNA binding), we ensure that tasks remain technology-independent. We provide examples of benchmarks and models implementing this framework, and anticipate that the community will contribute their own, leading to an ever-expanding and evolving set of models and evaluation tasks. This resource will accelerate genomics research by illuminating the best models for a given task, motivating novel functional genomic benchmarks, and providing a more nuanced understanding of model abilities.

bioinformatics↗

bioRxiv2025

Precision culturomics enabled by unlabeled single-cell morphology and Raman spectra

Selective enrichment of target bacteria from complex communities such as the human microbiome has remained a challenge. Here we report a solution based on morphology, Raman spectrometry and the Laser-Induced Forward Transfer technology, which works at microbial single cells, many generations before they appear as colonies. We develop a machine learning-based framework that enables species-level targeted sorting of single microbial cells from complex microbiome. We illustrate the utility of this approach in selecting for or against specific bacteria in fecal microbiome samples and the potential for quantifying the molecules expressed based on Raman spectra. Analysis of single-cell cultured genomes reveals that brief antibiotic use drives both pre-existing resistance and de novo mutations in the transpeptidase or efflux pumps of gut commensals, along with convergent evolution between different species. Our precision culturomics method should enable detailed morphological, metabolic, and genomic insights into variations in microbial phenotypes at the single-cell level for microbiome studies.

microbiology↗

bioRxiv2025

Establishing a Digital PCR-Based Reference Measurement Procedure for Monkeypox Virus: An Inter-laboratory Assessment and Standardization Study

The accurate detection of monkeypox virus (MPXV) is crucial for effective viral diagnosis and epidemic prevention. Currently, the field lacks standardized reference methods for MPXV quantification, as well as reliable pseudovirus reference materials (RMs) to ensure quality control throughout the detection process. To address this, we developed a droplet digital PCR (ddPCR) reference measurement procedure (RMP) for precise quantification of MPXV genes B6R, which demonstrates excellent performance with a wide dynamic range (11-1.24x104 copies/L), strong linearity (R2=0.9984), and a low limit of quantitation (11 copies/L, CV[≤]25%). Repeatability tests confirmed high precision, with inter-laboratory CVs <10% across nine labs using different dPCR platforms. recovery efficiencies for B6R and F3L were [~]69%, with uncertainties incorporated into final measurements. The homogeneity assessment showed good results (CV=2.94%), and stability studies confirmed the stability of the sample during -70 {degrees}C and short-term storage (4 {degrees}C/-20 {degrees}C). Mandels statistical data shows that the reproducibility between laboratories is consistent. This validated ddPCR RMP provides metrological traceability, ensuring reliable and comparable results for MPXV diagnosis and supporting public health efforts.

molecular biology↗

bioRxiv2025

Method for Extracellular Electrochemical Impedance Spectroscopy on Epithelia

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting techniques, and error quantification. The measurement can be performed in approximately one minute using instrumentation capable of galvanostatic sinusoidal signal processing (4 A amplitude, 2 Hz-50 kHz). All fits to the model have less than 10 {Omega} mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n=3) and bronchiolar epithelial samples (n=4), we measured TER 500-667 {Omega}.cm2 and 955-1034 {Omega}.cm2, within the expected range, TEC 3.65-4.10 F/cm2 and 1.07-1.10 F/cm2, and membrane ratios 18-22 and 1.9-2.2, respectively.

bioengineering↗