Biology subjects

Ho, M.

Publications and source records attributed to Ho, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Heparan Sulfate Controls Nanoscale Assembly of GPC3-Wnt Receptor Complexes

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is highly expressed in hepatocellular carcinoma and promotes tumor progression through Wnt3a/{beta}-catenin signaling. However, how the nanoscale organization of GPC3 at the cell surface controls signaling remains unclear. Here, we combined nano-resolution MINFLUX imaging, single-molecule tracking, and functional assays to define the spatial architecture and dynamics of GPC3 on hepatoma cells. We found that GPC3 exists as both single molecules and nanoscale clusters and switches between confined and free diffusions on the plasma membrane. Heparan sulfate (HS) chains create nanoscale corrals that limit GPC3 movement, whereas removal of HS increases diffusive heterogeneity and disrupts confinement. Wnt3a stimulation induces the formation of higher-order GPC3 assemblies and enhances {beta}-catenin signaling, while loss of HS markedly reduces this response. MINFLUX DNA-PAINT further revealed that HS chains orchestrate the spatial distribution of Wnt3a and promote its association with the Wnt receptor, Frizzled-1, an essential step for pathway activation. Collectively, these findings reveal that HS controls the nanoscale organization and dynamics of GPC3 to promote Wnt receptor assembly and efficient {beta}-catenin signaling in hepatoma cells.

cell biology↗

bioRxiv2026

Engineering CAR T Cells for Hepatocellular Carcinoma Recurrence after Liver Transplantation

Background & AimsLiver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR/Cas9-mediated FKBP1A disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with TRAC knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity. MethodsHuman T cells were edited using Cas9 ribonucleoprotein complexes targeting FKBP1A and TRAC, expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed in vitro. Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model. FKBP1A/TRAC double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period. ResultsTacrolimus suppressed wild-type CAR T cell function but not FKBP1A/TRAC double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and in vivo antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy. TRAC disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional FKBP1A/TRAC knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant. ConclusionsDual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs. Impact and implicationsO_LIEngineering armored GPC3-targeting CAR T cells for hepatocellular carcinoma in the post-transplant setting C_LIO_LIDemonstrating safety and therapeutic efficacy in the presence of immunosuppressive drugs in preclinical models C_LIO_LILeveraging mTOR inhibitor resistance and CD3 destabilization enables high-efficiency, high-purity selection of genetically modified T cells C_LIO_LIA modular cellular engineering platform enables utilization for alternative tumor targets and next-generation CAR T cells C_LI

immunology↗

bioRxiv2026

Allogeneic CRISPR-Engineered CAR-T Cells Drive Potent Antitumor Activity in Solid Tumors

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, in part due to variability in autologous T cells derived from heavily pretreated patients with advanced disease. To address these constraints, we developed an off-the-shelf allogeneic CAR-T platform using CRISPR-Cas9-mediated genome editing in T cells from healthy donors to enable targeted CAR insertion at the TRAC locus with concurrent disruption of B2M. Using adeno-associated virus (AAV) delivery, we designed CAR-T cells targeting glypican-2 (GPC2) and glypican-3 (GPC3), emerging antigens expressed in pediatric and adult solid tumors. Genome-edited allogeneic CAR-T cells exhibited potent, antigen-specific cytotoxicity across multiple tumor models. GPC2-directed allogeneic CAR-T cells demonstrated enhanced or comparable activity relative to conventional lentiviral CAR-T cells in neuroblastoma models and mediated tumor regression with prolonged survival in preclinical models. Notably, repeated dosing augmented antitumor efficacy without evidence of toxicity, supporting multi-dose regimens for solid tumors. Similarly, GPC3-targeted allogeneic CAR-T cells based on a single-domain antibody showed robust activity against hepatocellular carcinoma cells in vitro and in vivo. These findings establish a scalable, genome-engineered allogeneic CAR-T strategy with strong therapeutic potential and support the clinical development of off-the-shelf cell therapies for pediatric and adult solid tumors.

immunology↗

bioRxiv2026

Crystal structure and molecular dynamics simulations of rademikibart Fab-IL-4Rα complex reveal biochemical basis for next-generation potent IL-4Rα inhibition in type 2 allergic and inflammatory diseases

Rademikibart (CBP-201) is a human monoclonal antibody with higher binding affinity to IL-4R compared to dupilumab. Dupilumab is a first-generation interleukin-4 receptor alpha (IL-4R) inhibitor for treating IL-4R-dependent inflammatory disorders, including several dermatologic and respiratory conditions. Rademikibart, however, demonstrated better inhibition of STAT6 intracellular signaling in vitro and similar potency in inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation. To further characterize the molecular function of rademikibart and its differentiation from dupilumab, we determined the crystal structure of the rademikibart fragment antigen binding (Fab) bound to IL-4R at 2.71 [A] resolution and compared this to the 2.82 [A] resolution structure of dupilumab Fab bound to IL-4R. The rotation angle between dupilumab and rademikibart bound to IL-4R is 54.88{degrees}. This rotation enables the binding epitopes of rademikibart, but not dupilumab, on IL-4R to overlap more closely with the conserved binding interface naturally utilized by IL-4 and IL-13 cytokines. Molecular dynamics (MD) studies on rademikibart and dupilumab bound to IL-4R examined the stability of the complexes and effects of amino acid mutations on receptor complex formation. MD simulations demonstrated that the third interface loop (residues 145 to 153 in domain 2) of IL-4R interacts directly with rademikibart, which is absent in the dupilumab/IL-4R complex. This finding is confirmed by increased hydrogen bond interactions at the interface between rademikibart and IL-4R, demonstrating superior binding energy for rademikibart. Through analysis of the x-ray crystallography structures, MD-equilibrated structures, and computational point-mutation analysis of rademikibart, we identified residue Y50 and R55 of the light chain and R97, R99, and Y101 of the heavy chain of rademikibart as key residues interacting with IL-4Rs third interface loop. Our data provides a molecular and structural rationale for the enhanced IL-4R inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4R inhibitor.

biochemistry↗

bioRxiv2026

Enteroviral epitope mimicry enables NK cell-mediated targeting of ASPH in hepatocellular carcinoma

Cancer development is shaped by host-microbe interactions, including viral infections. While several viruses are established oncogenic drivers, their potential protective roles in cancer remain unclear. Here we identify a dominant antibody response to CE1, a consensus epitope of enterovirus and rhinovirus, that is associated with reduced hepatocellular carcinoma (HCC) incidence and mortality. Anti-CE1 antibodies selectively recognize HCC cells and mediate anti-tumor activity through NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Mechanistically, anti-CE1 antibodies cross-react with aspartate {beta}-hydroxylase (ASPH), with CE1-ASPH sequence homology underpinning tumor recognition and cytotoxicity. Clinically, ASPH is aberrantly upregulated in HCC and correlates with inferred NK cell-associated ADCC activity and improved survival in CE1-seropositive patients. Collectively, these findings reveal a mechanism by which antiviral humoral immunity confers cancer protection through molecular mimicry and highlight anti-CE1 immunity as a potential therapeutic strategy in HCC.

cancer biology↗

bioRxiv2026

Insights into tick-pathogen interactions - a single cell RNA sequencing approach of transcriptional changes during ehrlichial infection

Tick-borne diseases represent a significant threat to human and animal health worldwide. In the United States, the blacklegged tick, Ixodes scapularis (I. scapularis), serves as a competent vector for several bacterial pathogens, including Ehrlichia muris eauclairensis (EME). The I. scapularis embryonic cell line (ISE6) is a valuable tool for propagating tick-borne pathogens and studying tick-pathogen interactions. In this study, we examined the cellular complexity of ISE6 cells and their response to EME infection. Single-cell RNA sequencing revealed 15 distinct cell clusters present. Although ISE6 cells are heterogeneous, they do not display transcriptional similarity to any known tick tissues. Notably, this lack of similarity did not influence their susceptibility to EME infection. Our results demonstrated that EME infection induces time-dependent transcriptional changes in ISE6 cells: early infection is characterized by upregulation of genes associated with stress adaptation, mitochondrial function, and metabolic pathways, whereas late infection leads to broad downregulation of genes involved in the cell cycle, DNA replication, and cytoskeletal organization. These findings enhance our understanding of ehrlichial interactions with ISE6 cells and reinforce the utility of this cell line as a resource for isolating and propagating arthropod endosymbionts and tick-borne pathogens. IMPORTANCEThis study provides a single-cell resolution framework for interpreting tick cell line biology during infection with a medically relevant ehrlichial pathogen. Using scRNA-seq, we show that the I. scapularis embryonic-derived ISE6 cell line comprises multiple transcriptionally distinct cell states, yet these states do not map cleanly onto canonical tick tissue signatures, even when compared against a curated reference tissue atlas. Despite this heterogeneity, EME broadly infects ISE6 cell population, indicating that susceptibility is not restricted to a specific cell type. We further define a time-dependent arthropod vector response in which early infection is marked by activation of stress and metabolic adaptation response, followed by late-stage inhibition of key signaling, transcriptional, and proliferative pathways as bacterial burden increases. Together, these findings strengthen the biological interpretation of ISE6 as an in vitro model for tick-pathogen interactions and provide a resource for future mechanistic studies of ehrlichial persistence, replication, and vector competence.

molecular biology↗

bioRxiv2025

Multimodal Approach for Identification and Validation of Hepatocellular Carcinoma Targets for Radiotheranostics

Identifying tumor selective targets is critical for the development of precision diagnostic and therapeutic agents in oncology. Despite advances in precision oncology elsewhere, there are no FDA-approved hepatocellular carcinoma (HCC)-selective treatments. HCC is the most common type of liver cancer and accounts for significant morbidity and mortality worldwide. Here, we sought to integrate bulk (371 cases) and single cell RNA sequencing (scRNAseq, n=2 datasets, 34 cases, 102,956 cells) of patient samples to enrich for molecules that are overexpressed in HCC, which could serve as HCC-selective targets. To guide definitions of tumor and normal cell clusters with higher fidelity, we also imported a normal liver scRNAseq dataset. Using this integrated approach, we identified several HCC-selective plasma membrane molecules. To validate these targets, we performed immunohistochemical staining of HCC and normal tissue microarrays and confirmed HCC-selective staining of identified targets. Next, we verified the presence of these targets in several commercially available HCC cell lines by flow cytometry and western blot. Finally, we designed, engineered, and tested novel antibody-based positron emission tomography (immunoPET) agents to these targets in various murine models of liver cancer. Our findings confirm that we can leverage this multimodal approach to identify and validate of HCC-selective targets, which can be used to develop tumor-selective diagnostic and therapeutic radiopharmaceuticals, or radiotheranostics, and other precision oncology agents. One Sentence SummaryA multimodal pipeline defines and validates tumor-selective surface targets for radiotheranostic use in hepatocellular carcinoma.

cancer biology↗

bioRxiv2025

Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

The lack of disease-modifying treatments for schizophrenia necessitates the exploration of novel aspects of its pathophysiology, including innate immune mechanisms in the periphery. C4 protein activation, associated with the complement cascade of innate immunity, associates with symptoms and predicts outcomes. However, C4 protein activation does not coincide with expected changes to other proteins in the complement cascade, suggesting another source of C4 protein activation. Using a combination of fresh whole blood from ten anonymous donors and a large set of publicly available microarray data, we show, for the first time, that C4 protein is found and expressed primarily in neutrophils and monocytes. Then, we compared the correlation between C4 protein in neutrophils, classical monocytes, plasma and the number of C4A gene copies. We determined the number of C4A genes using digital droplet PCR, C4 protein in neutrophils (15 patients/21 controls) and plasma (30 patients/38 controls) using western blotting, and classical monocytes (30 patients/38 controls) using flow cytometry. We found a moderate positive correlation between the number of C4A gene copies and the amount of C4 protein only in neutrophils and only in the schizophrenia group (Spearmans rho, r = 0.64, p = 0.01, Cohens d [~]1.67). Our results indicate a convergence of innate immunity mechanisms associated with schizophrenia. This novel mechanism of innate immunity in schizophrenia deserves further study to determine whether it could be a useful drug target.

immunology↗

bioRxiv2025

Effect of cortisol on cortical organoids: Building a "stress in a dish" model system

Exposure to chronic stress and traumatic experiences impacts brain health and development, which may lead to Post Traumatic Stress Disorder (PTSD), other mental health conditions, or altered resilience. Although certain behavioral and social factors have been associated resilience, little is known about the cellular and genomic mechanisms contributing to resilience or developing PTSD. Here, we present a novel model system called "Stress-in-a-dish" (SIAD) to study the molecular signature of chronic and acute stress in differentiated cortical organoids. Derived from human induced Pluripotent Stem Cells (iPSCs), mature organoids responded to cortisol with differential expression of genes. Many genes were associated with expected corticosteroid pathways, and some have not been previously linked to PTSD. A previously unknown potential contribution of astrocytes to the etiology of stress responses was also found. Our results suggest a novel paradigm for studying stress in a dish that opens up new opportunities to understand the biological basis of PTSD and resilience.

genomics↗

bioRxiv2025

Colonization with Oxalobacter formigenes slows the progression of CKD and reduces cardiac remodeling in CKD

Accumulation of oxalate in patients with chronic kidney disease (CKD) is associated with CKD progression and increased risk of cardiac death. Whether reducing plasma or urine oxalate slows CKD progression and prevents cardiovascular complications remains unexplored. We colonized the intestines of control and CKD mice with Oxalobacter formigenes (Oxf), an oxalate-degrading microorganism. The mice were fed with the oxalate precursor hydroxyproline for 23 weeks at which time we assessed pathological changes in the kidney and heart. We demonstrate that Oxf reduces plasma oxalate (pOx) and creatinine levels, mitigates inflammation and fibrosis in the kidney, and reduces pathologic cardiac remodeling in the hearts of CKD mice. RNA-seq analysis of ventricular tissue of CKD mice reveals dysregulated expression of metabolic pathways while Oxf colonization reverses these changes. These findings demonstrate that oxalate accumulation plays a role not only in CKD progression but also in associated cardiovascular complications and suggest that strategies to reduce plasma oxalate levels may have therapeutic benefit. Translational statementChronic kidney disease (CKD) is a major health problem that can lead to kidney failure and which increases the risk of cardiovascular disease (CVD) mortality. Oxalate accumulation in advanced kidney disease contributes to further CKD progression and CVD complications. Intestinal colonization with Oxalobacter formigenes (Oxf) in a CKD animal model reduces plasma oxalate level and slows progression of both CKD and CVD. Strategies to reduce plasma oxalate levels may have therapeutic benefit in the setting of CKD.

microbiology↗

bioRxiv2025

Temporal mapping of the anti-tumor effects of nanobody-based MSLN.CAR-T cell therapy in metastatic solid tumors

Studies on the dynamic changes occurring in the tumor microenvironment (TME) following CAR-T cell therapy have been confounded by host lymphodepletion, multiple dosing and immunodeficient models. Here, a nanobody-based, mouse mesothelin-targeting CAR-T cell (A101) was developed, achieving effective primary tumor suppression, metastasis reduction, and improved survival after a single dose in immunocompetent, syngeneic mouse models without lymphodepletion. Temporal tumor profiling using RNA sequencing revealed initial downregulation of cell proliferation genes followed by upregulation of inflammation, epithelial-to-mesenchymal-transition (EMT) and extracellular matrix (ECM) modification genes in the CAR-T-treated tumors relative to mock-T-treated controls. This phenotype was reversed at a later timepoint which coincided with downregulation of immunosuppressive Cd274+ Lcn2+ neutrophils and upregulation of anti-tumor P2rx1+ Nrf2- neutrophils. At the same time, upregulation of Ccl2+ in fibroblasts and a more immunomodulatory macrophage phenotype was observed in CAR-T-treated tumors, indicating a tumor adaptation mechanism. This study demonstrates complex dynamic changes in the TME, and highlights time-dependent responses of solid tumors to CAR-T cell therapy. It further highlights Lcn2+ neutrophils and Ccl2+ fibroblasts as potential therapeutic targets for improving CAR-T cell anti-tumor efficacy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/640438v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@88ec4org.highwire.dtl.DTLVardef@1d4fa64org.highwire.dtl.DTLVardef@182e11aorg.highwire.dtl.DTLVardef@1889182_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Tumor responses to A101 CAR-T cell treatment. C_FIG

immunology↗

bioRxiv2024

Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1

Despite its importance in pathogenesis, the hematogenous dissemination pathway of B. burgdorferi is still largely uncharacterized. To probe the molecular details of transendothelial migration more easily, we studied this process using cultured primary or telomerase-immortalized human microvascular endothelial cells in a medium that maintains both the human cells and the spirochetes. In B. burgdorferi infected monolayers we observed [~]55% of wild-type spirochetes crossing the monolayer. Microscopic characterization revealed entrance points across the cellular surface rather than at cellular junctions, supporting a transcellular route. In support of this pathway, locking the endothelial junctions using a VE-PTP inhibitor did not reduce transendothelial migration. We also used inhibitors to block the most common endocytic pathways to elucidate effectors that might be involved in B. burgdorferi uptake and/or transmigration. Directly inhibiting Cdc42 reduced spirochete transmigration by impeding internalization. However, blocking Rac1 alone dramatically reduced transmigration and resulted in a concomitant increase in spirochete accumulation in the cell. Our combined results support that B. burgdorferi internalization is an intermediate step in the transendothelial migration process which requires both Cdc42 and Rac1; Cdc42 is needed for spirochete internalization while Rac1 is required for cellular egress. These are the first two host proteins implicated in B. burgdorferi transmigration across endothelial cells. IMPORTANCELyme borreliosis is caused by Borrelia burgdorferi and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manisfestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of B. burgdorferi across a monolayer of microvascular endothelial cells grown in culture. We show that this occurs by passage of the spirochetes directly through these cells rather than at cellular junctions and that internalization of B. burgdorferi is an intermediate step in the transmigration process. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.

microbiology↗

bioRxiv2023

Dynamic Responses of Striatal Cholinergic Interneurons Control the Extinction and Updating of Goal-Directed Learning

Striatal cholinergic interneurons (CINs) regulate behavioral flexibility, but their exact contribution to this process remains elusive. In this study, we report that extinction learning enhances acetylcholine (ACh) release. Mimicking this enhancement by optogenetically inducing CIN burst firing promotes extinction learning. CINs receive excitatory thalamic inputs, and we observed that extinction training augmented thalamic activity. Optogenetically stimulating these thalamic inputs caused CIN burst firing and enhanced ACh release, strengthening extinction learning. Notably, CIN burst firing is usually followed by a pause in firing. We found that disrupting this pause through continuous optogenetic stimulation reversibly impaired the updating of goal-directed behaviors. Furthermore, excessive alcohol consumption reduced thalamus-induced burst-pause firing in CINs and impaired the reversal of goal-directed learning. In summary, thalamic-driven CIN burst firing promotes extinction learning, while the pause is pivotal for reversing goal-directed behavior, a process impacted by excessive alcohol. These findings shed light on how CINs dynamic responses affect behavioral flexibility. HighlightsH1. Burst firing of CINs promotes extinction learning H2. Thalamic-CIN excitation enhances extinction learning H3. Pause of CIN is critical for the reversal of goal-directed learning H4. Chronic alcohol consumption reduces the burst-pause of CINs and impairs the reversal of goal-directed learning.

neuroscience↗

bioRxiv2023

Tumor-Associated Macrophages Expand Chemoresistant, Ovarian Cancer Stem-Like Cells

The persistence of ovarian cancer stem-like cells (OvCSCs) after chemotherapy resistance has been implicated in relapse. However, the ability of these relatively quiescent cells to produce the robust tumor regrowth necessary for relapse remains an enigma. Since normal stem cells exist in a niche, and tumor-associated macrophages (TAMs) are the highest abundance immune cell within ovarian tumors, we hypothesized that TAMs may influence OvCSC proliferation. To test this, we optimized OvCSC enrichment by sphere culture and in vitro polarization of monocytes to a TAM-like M2 phenotype. Using cocultures that permitted the exchange of only soluble factors, we found that M2 macrophages increased the proliferation of sphere cells. Longer-term exposure (5-7 days) to soluble TAM factors led to retention of some stem cell features by OvCSCs but loss of others, suggesting that TAMs may support an intermediate stemness phenotype in OvCSCs. Although TAM coculture decreased the percentage of OvCSCs surviving chemotherapy, it increased the overall number. We therefore sought to determine the influence of this interaction on chemotherapy efficacy in vivo and found that inhibiting macrophages improved chemotherapy response. Comparing the gene expression changes in OvCSCs cocultured with TAMs to publicly available patient data identified 34 genes upregulated in OvCSCs by exposure to soluble TAM factors whose expression correlates with outcome. Overall, these data suggest that TAMs may influence OvCSC proliferation and impact therapeutic response.

cancer biology↗

bioRxiv2023

The ENCODE Uniform Analysis Pipelines

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses. Database URL: https://www.encodeproject.org/

bioinformatics↗

bioRxiv2023

Structural basis for differential p19 targeting by IL-23 biologics

BackgroundIL-23 is central to the pathogenesis of psoriasis, and is structurally comprised of p19 and p40 subunits. "Targeted" IL-23 inhibitors risankizumab, tildrakizumab, and guselkumab differ mechanistically from ustekinumab because they bind p19, whereas ustekinumab binds p40; however, a knowledge gap exists regarding the structural composition of their epitopes and how these molecular properties relate to their clinical efficacy. ObjectivesTo characterize and differentiate the structural epitopes of the IL-23 inhibitors risankizumab, guselkumab, tildrakinumab, and ustekinumab, and correlate their molecular characteristics with clinical response in plaque psoriasis therapy. MethodsWe utilized epitope data derived from hydrogen-deuterium exchange studies for risankizumab, tildrakizumab, and guselkumab, and crystallographic data for ustekinumab to map drug epitope locations, hydrophobicity, and surface charge onto the IL-23 molecular surface (Protein Data Bank ID Code 3D87) using UCSF Chimera. PDBePISA was used to calculate solvent accessible surface area (SASA). Epitope composition was determined by classifying residues as acidic, basic, polar, or hydrophobic and calculating their contribution to epitope SASA. Linear regression and analysis of variance was performed. ResultsAll the p19-specific inhibitor epitopes differ in location and size, with risankizumab and guselkumab having large epitope surface areas (SA), and tildrakizumab and ustekinumab having smaller SA. The tildrakizumab epitope was mostly hydrophobic (56%), while guselkumab, risankizumab, and ustekinumab epitopes displayed >50% non-hydrophobic residues. Risankizumab and ustekinumab exhibited acidic surface charges, while tildrakizumab and guselkumab were net neutral. Each inhibitor binds an epitope with a unique size and composition, and with mostly distinct locations except for a 10-residue overlap region that lies outside of the IL-23 receptor epitope. We observed a strong correlation between epitope SA and PASI-90 rates (R2 = 0.9969, p = 0.0016), as well as between epitope SA and KD (R2 = 0.9772, p = 0.0115). In contrast, we found that total epitope hydrophobicity, polarity, and charge content do not correlate with clinical efficacy. ConclusionsStructural analysis of IL-23 inhibitor epitopes reveals strong association between epitope SA and early drug efficacy in plaque psoriasis therapy, exemplifying how molecular data can explain clinical observations, inform future innovation, and help clinicians in specific drug selection for patients.

molecular biology↗

bioRxiv2023

Elevated Glucocorticoid alters the trajectory of hypothalamic development and function

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analysed the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and longevity. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic development trajectory, a process crucial for health across the life-course.

neuroscience↗

bioRxiv2022

Monitoring Non-Specific Adsorption at Solid-Liquid Interfaces by Supercritical Angle Fluorescence Microscopy

Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been used extensively to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored. Here we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface phenomena thereby greatly expanding the range of potential applications of this tool. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of BSA and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixture with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 seconds of addition. The adsorption rate constant (ka) and half-life of BSA adsorption on Teflon-AF were determined to be 0.2093{+/-}0.002 s-1 and 3.312{+/-}0.032 s respectively using a pseudo-first-order adsorption equation.

biophysics↗