Biology subjects

Wang, J.

Publications and source records attributed to Wang, J..

At least 703 records · Page 39Linked to original sources

bioRxiv2025

Multimodal retinal imaging by visible light optical coherence tomography and phosphorescence lifetime ophthalmoscopy in the mouse eye

SignificanceOxygen metabolism is important to retinal disease development, but current imaging methods face challenges in resolution, throughput, and depth sectioning to spatially map microvascular oxygen. AimTo develop a multimodal system capable of simultaneous phosphorescence lifetime imaging scanning laser ophthalmoscopy (PLIM-SLO) and visible light optical coherence tomography (VIS-OCT) to capture capillary-level oxygen partial pressure (pO2) and structural volumes in rodents. ApproachC57BL/6 mice were imaged by VIS-OCT with high-definition (10 kHz raster) and Doppler (100 kHz circular) protocols. Phosphorescent probe Oxyphor 2P was retro-orbitally injected to enable intravascular PLIM-SLO imaging (200 {micro}s pixel dwell time), while a tunable lens was used to adjust the focal depth. The extracted phosphorescence lifetimes were used for pO2 calculation. Simultaneous imaging utilized a shared imaging path and synchronized data collection. ResultsVIS-OCT images revealed detailed anatomy and Doppler shifts, while PLIM-SLO provided capillary pO2 at multiple depths. A hemoglobin oxygen dissociation curve related retinal arterial pO2 to systemic oxygen saturation as inhaled oxygen was varied. Registered simultaneous images were captured and pO2 was empirically adjusted for the combined excitation. ConclusionsDetailed anatomical structures and capillary pO2 levels can be simultaneously imaged, providing a useful tool to study oxygen metabolism in rodent disease models.

bioengineering↗

bioRxiv2025

Prenatal Alcohol Exposure Impairs Striatal Cholinergic Function and Cognitive Flexibility in Adult Offspring

Fetal Alcohol Spectrum Disorder (FASD), caused by prenatal alcohol exposure (PAE), is characterized by significant cognitive impairments, including reduced cognitive flexibility. Despite the critical role of cholinergic interneurons (CINs) in the dorsomedial striatum (DMS) for cognitive and behavioral flexibility, their contribution to neurobehavioral deficits in FASD remains unclear. To address this gap, this research explored the impact of PAE on CIN populations and activity, cognitive flexibility, and compulsive drinking behaviors in adult offspring. Using ChAT-Cre;Ai14-tdTomato mice combined with ChAT staining, we found substantial reductions in CIN number within the striatum of adult PAE offspring. Functional assessments revealed that PAE markedly decreased CIN firing activity and reduced acetylcholine (ACh) release in the DMS, as measured by electrophysiology recordings and live-tissue confocal imaging using a genetically encoded ACh sensor. Behaviorally, PAE-exposed offspring exhibited a significant deficit in adapting to reversed action-outcome contingencies despite intact initial learning capabilities. Moreover, PAE-exposed mice exhibited compulsive alcohol drinking behavior, characterized by elevated consumption and preference for quinine-adulterated alcohol. These findings collectively highlight the critical role of impaired cholinergic signaling in the cognitive and behavioral deficits observed following PAE. Understanding this cholinergic dysfunction provides valuable insights necessary for developing targeted interventions aimed at mitigating cognitive and behavioral consequences associated with FASD. HighlightsPAE reduces the number of CINs, their firing rate and ACh release in the DMS. PAE impaired instrumental reversal learning, known to be mediated by CINs. PAE increased compulsive-like drinking of quinine adulterated alcohol.

neuroscience↗

bioRxiv2025

An Adipose mTORC1 Effector Links Nutrient-induced Ribosomal Protein Translation to Lifespan in Drosophila

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to orchestrate metabolic processes critical for physiological homeostasis and organismal ageing1. While mTORC1 preferentially regulates the translation of 5'-terminal oligopyrimidine (TOP) motif-containing mRNAs that predominantly encode ribosomal proteins (RPs) via the translational repressor 4E-BP 2, this mTORC1 function is resistant to rapamycin inhibition 3. TOP mRNAs are exceptionally abundant, thus imposing a major translational burden on cells; yet how their translation is physiologically tuned and linked to longevity remain unexplored. Here we identify Lsp2, originally known as a storage protein 4, as an adipose effector of mTORC1 that modulates lifespan in Drosophila. Lsp2 is induced by essential amino acids (EAAs) via mTORC1. Genetic ablation of Lsp2 to blunt organismal response to protein diets drives robust lifespan extension without compromising key life-history traits such as reproduction. Translatomic profiling reveals that loss of Lsp2 selectively reduces global TOP mRNA translation in a 4E-BP-dependent manner, thereby extending lifespan via a mechanism distinct from rapamycin inhibition that fails to reduce 4E-BP phosphorylation in vivo. Finally, Lsp2 adipokine promotes 4E-BP phosphorylation and acts systemically across tissues to shape the lifespan responses to dietary protein. Collectively, our findings establish Lsp2 as a novel translational regulator of TOP genes that mechanistically couples physiological ribosomal protein synthesis with organismal longevity.

physiology↗

bioRxiv2025

How Good is AlphaFold3 at Ranking Drug Binding Affinities?

Accurate affinity ranking of small molecules is pivotal for drug discovery. We investigate whether structure prediction models like AlphaFold3, pretrained on protein-ligand interactions, can address this task. Zero-shot evaluation of Protenix (an AlphaFold3-like model) demonstrates superior prioritization of active compounds over conventional scoring functions and state-of-the-art deep learning models. By further fine-tuning Protenix on structure-agnostic protein-ligand bioactivity data from ChEMBL and BindingDB, we develop AlphaRank that predicts pairwise affinity relationships. AlphaRank achieves prediction accuracy comparable to computationally intensive free energy perturbation (FEP+) workflows on standard benchmarks, while requiring substantially less computational resources. Our findings highlight the emergent potential of AlphaFold3-derived models in affinity ranking tasks and emphasize the necessity for targeted methodological exploration to fully harness their capabilities in drug discovery applications.

bioinformatics↗

bioRxiv2025

A Diffusion-Based Framework for Designing Molecules in Flexible Protein Pockets

The design of molecules for flexible protein pockets represents a significant challenge in structure-based drug discovery, as proteins often undergo conformational changes upon ligand binding. While deep learning-based approaches have shown promise in molecular generation, they typically treat protein pockets as rigid structures, limiting their ability to capture the dynamic nature of protein-ligand interactions. Here, we introduce YuelDesign, a novel diffusion-based framework specifically developed to address this challenge. YuelDesign employs a new protein encoding scheme with a fully connected graph representation to encode protein pocket flexibility, a systematic denoising process that refines both atomic properties and coordinates, and a specialized bond reconstruction module tailored for de novo generated molecules. Our results demonstrate that YuelDesign generates molecules with favorable drug-likeness and low synthetic complexity. The generated molecules also exhibit diverse chemical functional groups, including some not even present in the training set. Redocking analysis reveals that the generated molecules exhibit docking energies comparable to native ligands. Additionally, a detailed analysis of the denoising process shows how the model systematically refines molecular structures through atom type transitions, bond dynamics, and conformational adjustments. Overall, YuelDesign presents a versatile framework for generating novel molecules tailored to flexible protein pockets, with promising implications for drug discovery applications.

bioinformatics↗

bioRxiv2025

RNA-seq of seven non-neoplastic/inflammatory lymphadenitis entities identify potential molecular diagnostic markers

Lymphadenopathy is a common mode of presentation for various disorders. While malignancy must be considered, there is a broad spectrum of causes that are infectious or of autoimmune origin. Clinical testing and morphological assessment are required to distinguish between neoplasms and benign pathologies. However, many causes of lymphadenopathy have similar histological presentations, making definitive diagnoses and categorization challenging. While DNA sequencing have enabled the accurate diagnosis of certain disorders, such as the genetic origin of ALPS (autoimmune lymphoproliferative syndrome), other causes of lymphadenopathy such as benign sinus histiocytosis, or Rosai-Dorfman disease (RDD), cannot be diagnosed with current technologies. Likewise, several modes of infection, including Toxoplasmosis gondii, Epstein-Barr virus (EBV), and Bartonella henselae (Cat-scratch disease) require further investigation. RNA sequencing enables the assessment of differentially expressed genes (DEG) that reveal up or downregulated pathways. These DEG signatures and pathways can help differentiate lymphadenopathies that appear similar histologically. In this study, RNA sequencing of 46 cases of lymphadenopathies were performed and analyzed. ALPS and RDD cases provided confirmation that RNA sequencing can identified expected gene expression and activated pathways. Our results showed the unique activation of the interferon pathway was identified in sinus histiocytosis cases. Through advances in RNA sequencing, potential genetic signatures can be discovered in a gamut of varying conditions.

molecular biology↗

bioRxiv2025

Evaluation of visual performance for RCS rats: A tailor-made OMR setup via DeepLabCut and PsychoPy with a customized algorithm

In this study, we developed an optimized optomotor response (OMR) setup for rats based on existing commercial systems and introduced a novel method that integrates DeepLabCut, PsychoPy, and custom algorithms to automate OMR performance evaluation in rats. Our primary findings revealed the optimal spatial frequency (SF) and grating velocity for pigmented Royal College of Surgeons (RCS) and wild-type (WT) rats. The RCS rats, a model for retinal degeneration, displayed normal OMR performance at postnatal days 30 (P30) and 50 (P50), but a significant decline was observed at 3 months. In most rats aged 6 and 12 months, the percentage of correct responses (PoC) was 50%, indicating blindness. The high efficiency and reproducibility of our non-invasive OMR test, along with its suitability for post-surgical and retinal degeneration models, underscores its potential for broad applications in visual performance evaluation.

neuroscience↗

bioRxiv2025

FOXM1 Modulation Alleviates Epithelial Remodeling and Inflammation in Eosinophilic Esophagitis

BackgroundEosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal epithelial remodeling, barrier dysfunction, and inflammation. Despite histologic remission, molecular and structural changes in the epithelium persist, contributing to ongoing symptoms and relapse. The transcription factor FOXM1 has been shown to be a key regulator of epithelial proliferation and inflammation in allergic asthma. ObjectiveTo investigate the role of FOXM1 in epithelial disruption in EoE and to evaluate the therapeutic potential of FOXM1 inhibition. DesignFOXM1 expression was analyzed in human esophageal biopsies, patient-derived organoids, and murine EoE models. IL-13 stimulation was used to model EoE in vitro. The effects of FOXM1 inhibition via the small molecule RCM-1 and siRNA-mediated knockdown were assessed by histology, gene expression profiling, organoid formation rates, and barrier integrity assays. RNA sequencing and chromatin immunoprecipitation were performed to elucidate molecular mechanisms. ResultsFOXM1 was significantly upregulated in patients with active EoE and localized to the basal epithelium. IL-13 increased FOXM1 expression, which impaired epithelial differentiation and enhanced basal cell hyperplasia. FOXM1 inhibition restored differentiation markers, reduced basal hyperplasia, and improved barrier function. In murine models, RCM-1 ameliorated epithelial changes and decreased eosinophil infiltration. Mechanistically, FOXM1 directly regulated cell cycle gene, CCNB1, which was upregulated in EoE and downregulated upon FOXM1 inhibition. FOXM1 expression was driven by an IL-13-PI3K/AKT axis. ConclusionFOXM1 plays a pivotal role in epithelial disruption in EoE by driving proliferation and impairing differentiation. Targeting FOXM1 restores epithelial homeostasis, mitigates inflammation, and offers a novel therapeutic approach for EoE. Key MessagesWhat is already known on this topic: Eosinophilic esophagitis is marked by epithelial remodeling and barrier dysfunction driven by Th2 inflammation. Despite remission, molecular and histologic changes in the esophageal epithelium persist, contributing to symptoms and relapse. The mechanisms underlying this epithelial dysregulation remain poorly understood. What this study adds: This study identifies FOXM1 as a key transcriptional regulator of epithelial disruption in EoE, demonstrating that FOXM1 inhibition restores epithelial differentiation, reduces basal cell hyperplasia, improves barrier integrity, and mitigates inflammation. How this study might affect research, practice, or policy: Targeting FOXM1 offers a novel therapeutic strategy to restore epithelial homeostasis and reduce inflammation in EoE. This dual approach, addressing both epithelial and immune dysregulation, may guide future therapeutic development and improve patient outcomes.

molecular biology↗

bioRxiv2025

OPTRACE: Optical Imaging Guided Transplantation and Tracking of Cells in the Mouse Brain

Intracerebral cell transplantation holds great promise for treating stroke and other neurological disorders, but progress is hindered by challenges in precise cell delivery and limited methods for monitoring post-engraftment dynamics. To address these limitations, we introduce OPTRACE (OPtical imaging-guided Transplantation and tRAcking of CElls), a two-step framework that integrates precision delivery with longitudinal in vivo tracking to advance cell therapy. During transplantation, OPTRACE enables real-time visualization using cost-effective translucent glass micropipettes, guided by predictive modeling to optimize delivery parameters. A novel pulse-elevation injection technique further enhances the precision of superficial cortical engraftment. Following transplantation, multicolor labeling combined with two-photon fluorescence microscopy permits longitudinal single-cell tracking, revealing host microglial responses, and altered neuronal calcium signaling at the graft interface. By unifying high-resolution delivery and tracking strategies, OPTRACE provides a powerful platform for elucidating engraftment biology and accelerating the development of effective cell-based interventions for neurological diseases.

bioengineering↗

bioRxiv2025

IL-17A-driven Macrophage Disorder Promotes Plaque Instability in Psoriatic Atherosclerosis

Psoriasis is a common chronic inflammatory skin disease that predisposes individuals to cardiovascular diseases such as atherosclerosis. However, the underlying mechanisms linking these conditions have not yet been fully elucidated. Recent studies have indicated that the inflammatory cytokine IL-17A plays a significant role in both diseases. The aim of this study was to investigate how psoriasis affects the stability of atherosclerotic plaques via the IL-17A/IL-17RA pathway. We performed single-cell sequencing on patients with both psoriasis and atherosclerosis to analyze changes in the immune microenvironment within their carotid plaques and psoriatic lesions. We found that IL-17A significantly upregulated the expression of pro-inflammatory factors (e.g., CXCL8) in macrophages of psoriatic atherosclerosis patients, leading to weakening of the fibrous cap structure of plaques. Additionally, IL-17A further exacerbated plaque instability by interfering with macrophage lipid metabolism, particularly sphingolipid metabolism. Finally, using in vitro experiments and animal models, we demonstrated that IL-17A enhances pro-inflammatory and metabolic abnormalities in macrophages by downregulating PPAR{gamma}. Therapeutic strategies targeting IL-17A may contribute to the simultaneous mitigation of psoriasis and atherosclerosis-associated cardiovascular risk, providing new directions for clinical intervention.

cell biology↗

bioRxiv2025

Hepatocyte-specific CLSTN3B ablation impairs lipid droplet maturation and alleviates diet-induced steatohepatitis in mice

Excessive lipid accumulation in hepatocytes, a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), can lead to progressive liver damage. Understanding the molecular mechanisms governing lipid storage in hepatocytes is essential for identifying therapeutic targets to halt MASLD progression. Here, we show a pivotal role for the protein calsyntenin 3{beta} (CLSTN3B) in promoting lipid droplet (LD) maturation and lipid storage in hepatocytes. Previously characterized as an endoplasmic reticulum (ER)-LD contact protein that facilitates LD maturation in adipocytes, we now show that CLSTN3B expression is strongly induced in mouse hepatocytes by peroxisome proliferator-activated receptor gamma (PPAR{gamma}) in response to dietary caloric excess. Hepatocyte-specific deletion of CLSTN3B in mice significantly increases energy expenditure, reduces metabolic efficiency, and protects against diet-induced hepatic steatosis and fibrosis. Mechanistically, CLSTN3B deficiency causes reduced LD phospholipid coverage and increased lipase recruitment. This results in enhanced fatty acid oxidation driven by a futile cycle of lipolysis and re-esterification. Notably, human clinical data reveal a positive correlation between hepatic CLSTN3B expression and MASLD severity and progression, emphasizing its relevance to human disease. Together, our findings establish CLSTN3B as a key regulator of hepatocyte lipid storage and metabolic efficiency and highlight its potential as a therapeutic target in MASLD.

physiology↗

bioRxiv2025

PLK1-mediated phosphorylation of PHGDH reprograms serine metabolism in advanced prostate cancer

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet their increased biosynthetic and energetic demands. While cells possess the capacity for de novo serine biosynthesis, most transformed cancer cells heavily depend on exogenous serine uptake to sustain their growth, yet the regulatory mechanisms driving this metabolic dependency remain poorly understood. Here, we uncover a novel mechanism by which Polo-like kinase 1 (PLK1), often overexpressed in prostate cancer, orchestrates a metabolic shift in serine and lipid metabolism through the phosphorylation of phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of the serine synthesis pathway (SSP). We demonstrate that PLK1 phosphorylates PHGDH at three specific sites (S512, S513, S517), leading to a marked reduction in its protein level and enzymatic activity. This downregulation of SSP forces cancer cells to increase their reliance on exogenous serine uptake via the ASCT2 transporter, which, in turn, fuels the biosynthesis of lipids, including sphingolipids essential for tumor growth and survival. Targeting the SSP, serine uptake, or downstream lipid biosynthetic pathways may offer promising therapeutic avenues in PLK1-high advanced cancers.

cancer biology↗

bioRxiv2025

Spatial Biology and Organoid Technologies Reveal a Potential Therapy-Resistant Cancer Stem Cell Population in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with a 5-year survival rate of less than 10%. Chemotherapy is the current standard-of-care (SOC) for advanced PDAC; however, treatment resistance driven by a complex and immunosuppressive tumor microenvironment (TME) limits its effectiveness. To address mechanisms of resistance, we studied cabozantinib (cabo), a multi-kinase inhibitor approved for several solid tumors. Cabo has shown promise in preclinical PDAC models and clinical trials, particularly when combined with immunotherapy, but its mechanisms of action within the human PDAC TME and its potential to overcome therapy resistance remain unclear. Using CosMx Spatial Molecular Imaging (CosMx SMI) and Orion Multiplex Immunofluorescence (MxIF), we analyzed PDAC tissues collected after first line systemic chemotherapy and the Whipple surgical procedure, as well as from liver or lung metastatic sites from patients with PDAC. To functionally model the TME, we established matched patient-derived organoid (PDO) co-cultures harboring cancer associated fibroblasts (CAFs), and autologous immune cells (IMM) (PDO/CAF/IMM). These models were used to evaluate the effects of cabo and pembrolizumab, an anti-PD1 immune checkpoint inhibitor, with benchmarking of findings to the patients TME. Spatial analysis of post-chemotherapy and metastatic PDAC tissues revealed heterogeneous cellular neighborhoods within the TME, including enrichment of Schwann cells, CAFs, T regulatory cells, and cancer stem cells (CSCs). Distinct niches were observed in metastatic liver tissues, characterized by mesenchymal stem cells, fibroblasts, including myCAFs and iCAFs, and CSCs expressing CD44 and TROP2. In PDO/CAF/IMM co-cultures, treatment with cabo in combination with pembrolizumab enhanced cancer cell death by depleting myeloid-derived suppressor cells (MDSCs) and promoted cytotoxic T lymphocyte proliferation. Across both patient tissues and treated co-cultures, a persistent SOC-resistant cancer stem cell population emerged that expressed CD44 variant 9 (CD44v9). Taken together, these integrative spatial and organoid-based studies demonstrate that cabo can remodel the PDAC TME and potentiate PD-1 immunotherapy in preclinical models, while resistance associates with a CD44v9+ CSC population, revealing a potential therapeutic target.

cancer biology↗

bioRxiv2025

AttentionAML: An Attention-based Deep Learning Framework for Accurate Molecular Categorization of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy defined by aberrant clonal expansion of abnormal myeloid progenitor cells. Characterized by morphological, molecular, and genetic alterations, AML encompasses multiple distinct subtypes that would exhibit subtype-specific responses to treatment and prognosis, underscoring the critical need of accurately identifying AML subtypes for effective clinical management and tailored therapeutic approaches. Traditional wet lab approaches such as immunophenotyping, cytogenetic analysis, morphological analysis, or molecular profiling to identify AML subtypes are labor-intensive, costly, and time-consuming. To address these challenges, we propose AttentionAML, a novel attention-based deep learning framework for accurately categorizing AML subtypes based on transcriptomic profiling only. Benchmarking tests based on 1,661 AML patients suggested that AttentionAML outperformed state-of-the-art methods across all evaluated metrics (accuracy: 0.96, precision: 0.96, recall of 0.96, F1-score: 0.96, and Matthews correlation coefficient: 0.96). Furthermore, we also demonstrated the superiority of AttentionAML over conventional approaches in terms of AML patient clustering visualization and subtype-specific gene marker characterization. We believe AttentionAML will bring remarkable positive impacts on downstream AML risk stratification and personalized treatment design. To enhance its impact, a user-friendly Python package implementing AttentionAML is publicly available at https://github.com/wan-mlab/AttentionAML.

bioinformatics↗

bioRxiv2025

Metabolic and ribosomal dysregulation drive developmental arrest of Schistosoma japonicum in non-permissive rats

Parasitic flatworms of the genus Schistosoma infect a wide range of definitive hosts, which are categorized as permissive or non-permissive based on their capacity to support parasite development. Unlike permissive hosts such as mice, Schistosoma japonicum undergoes pronounced developmental arrest in non-permissive hosts like rats; however, the molecular mechanisms underlying this phenomenon remain largely unclear. In this study, we conducted a comprehensive morphological and transcriptomic comparison of S. japonicum derived from mouse and rat hosts at multiple time points post-infection, identifying the period between 12 and 24 days post-infection (dpi) as a critical window for developmental arrest in parasites from rats. Utilizing single-cell RNA sequencing, we constructed a high-resolution cellular atlas of S. japonicum at 14 and 24 dpi from both host species, revealing host-dependent alterations in cell composition and transcriptional programs during parasite development. Functional analyses further demonstrated that impairments in mitochondrial function and anaerobic glycolysis contribute to the developmental arrest observed in parasites from rats. Additionally, downregulation of ribosomal genes was associated with reduced protein synthesis and impaired cell proliferation. Together, these findings suggest that disruptions in energy metabolism and ribosomal function are key drivers of developmental arrest in non-permissive hosts. This study provides novel, parasite-centered insights into host permissiveness and identifies potential molecular targets for schistosomiasis control strategies.

developmental biology↗

bioRxiv2025

Development of noninvasive imaging to measure spontaneous pain in mice

Objectively measuring pain in laboratory animals is essential for pain research and analgesic development. Despite the development of various behavioral tests to measure evoked pain in animal models, measuring spontaneous pain remains challenging. To address this unmet need, we developed a novel imaging approach to detect spontaneous nociception in animal pain models. To do this, we generated a Bacterial Artificial Chromosomes transgenic mouse that expresses Redquorin under the murine synapsin 1 promoter. Redquorin is a fusion protein consisting of chimera and a 2x tandem dimer Tomato Aequorin (tdTA), which emits long wavelength bioluminescence from activated neurons in the presence of coelenterazine. This luminescence can penetrate tissues and form a projected image on the body surface that can be detected with a spectrum In Vivo Imaging System, thus creating a Nociceptive Neuronal Activity Imaging mouse. We used the tdTA mice to image bioluminescence in the spinal regions as a surrogate of spontaneous pain induced by capsaicin, the HIV-1 envelope glycoprotein gp120, and spinal nerve ligation. Results show that Redquorin-emitted bioluminescence is a sensitive optical surrogate to measure spontaneous pain. This approach offers a new method to measure spontaneous pain in animal models for basic and translational research.

neuroscience↗

bioRxiv2025

Allosteric Modulation of MIF-2 Structure, Catalysis, and Biological Signaling via Cysteine Residues and a Small Molecule, Ebselen

The macrophage migration inhibitory factor (MIF) family of cytokines comprised of the MIF and D-dopachrome tautomerase (or MIF-2) paralogs share identical tertiary and quaternary structures that contribute to their overlapping enzymatic and signaling activities. Recent investigations of MIF and MIF-2 have shown them to possess N-to-C-terminal allosteric crosstalk, but despite the similarity of this "allosteric pathway," its regulation of MIF and MIF-2 is not identical. Thus, structure alone does not preserve the precise allosteric mechanism and additional residues that modulate MIF and MIF-2 allosteric function must be characterized. Cysteines have been identified as allosteric switches for the same biochemical functions of MIF and small molecules targeting its N-terminal enzymatic site have affected the structure of three proximal cysteines. Ebselen is a compound that forms covalent selenylsulfide bonds with MIF cysteines and is hypothesized to destabilize and dissociate the MIF trimer into monomers. Ebselen-bound MIF also displays little-to-no catalysis or biological signaling. However, it is unclear whether Ebselen similarly affects the MIF-2 paralog, despite MIF-2 containing two related cysteines (MIF contains three). We used mutagenesis, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, in vitro and in vivo biochemistry to investigate the mechanism of Ebselen as an allosteric modulator of MIF-2 via its cysteines. Our findings suggest that Ebselen partially disrupts the MIF-2 homotrimer, though the overall population of such a structure is <35%, even on the timescale of many hours. Ebselen does attenuate the biological functions of MIF-2 and solution structural biology captures the conformational transitions preceding the destabilized MIF-2 trimer. Significance StatementThe nearly identical MIF and MIF-2 structures have been recently shown to contain an N-to-C-terminal crosstalk between non-overlapping functional sites in vitro and in vivo. Small molecule inhibitors designed as therapeutics for the MIF superfamily have primarily targeted the N-terminal active site, while very few allosteric molecules have been reported. Ebselen, however, destabilizes MIF by covalently modifying its distal Cys80 residue and disrupting its obligate trimeric assembly. This unique mechanism has never been evaluated in the MIF-2 paralog, and this study reports the structural, dynamic, and functional impact of Ebselen on MIF-2. We define the essential structural hallmarks preceding trimer dissociation and reveal that Ebselen inhibits MIF-2 function before trimer disruption, leading to destabilized monomer-monomer interfaces that slowly degrade the quaternary assembly.

biophysics↗

bioRxiv2025

An aphid-resistant plant metabolite as a candidate aphicide: Insight into the bioactivity and action mode of betulin against aphids

Pest-resistant plants usually utilize secondary metabolites to cope with insect infestation. Betulin, a key bioactive compound in aphid-resistant wild peach, possesses promising applications in crop protection. Here, betulin, in both greenhouse and field experiments, displayed excellent control efficacy against Myzus persicae. RNA-seq, qRT-PCR, and western blotting revealed that betulin significantly inhibited the expression of MpGABR (encoding a GABAA receptor). Besides, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, MST (microscale thermophoresis) and voltage-clamp assays indicated that betulin bound to MpGABR (Kd = 2.24 {micro}M) and acted as an inhibitor of MpGABR. Molecular docking, mutagenesis and genome editing suggested that THR228 is a critical and highly conserved site in MpGABR that betulin binds to specifically, causing aphid death. Overall, the activity of betulin depends on specific targeting and inhibition of MpGABR. Elucidating the mechanism of action of this peach-derived insecticide may offer a sustainable green strategy for aphid control.

molecular biology↗