Biology subjects

Dong, J.

Publications and source records attributed to Dong, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Ultrashort optical-pin excitation for scattering brain imaging

Imaging neural structures deep in brain tissue is central to understanding brain function, yet remains fundamentally limited by strong optical scattering and the requirement for accurate three-dimensional (3D) optical sectioning. Laser-scanning microscopy is a promising technique for brain imaging; however, maintaining excitation focus integrity in scattering media while preserving axial confinement poses a persistent photonic challenge. Here we introduce the optical pin, an ultrashort excitation regime engineered at the angular-spectrum level to address this limitation. By broadening the transverse angular bandwidth of a Bessel-type field while preserving its conical momentum-space architecture, the optical pin introduces a controlled longitudinal wave-vector spread that compresses the axial interference length to the micrometer scale, restoring Gaussian-like sectioning without sacrificing multi-angle interference. This excitation design yields substantially enhanced imaging performance, including [~]1.5-fold contrast improvement and [~]2.6-fold increased robustness to scattering. We validate the approach across transparent, scattering, and biological specimens, including bead phantoms, C. elegans, and mouse brain tissue. As a system-level excitation strategy, the optical pin is readily compatible with existing laser-scanning microscopy platforms and is particularly suited for scattering-limited brain imaging.

neuroscience↗

bioRxiv2025

Mechanical Disturbance of Trichomes Enhances Cd2+ Uptake via Malate-Mediated Stomatal Opening in Arabidopsis

Phytoremediation of cadmium-contaminated soils relies on efficient metal uptake and translocation in plants. We previously found that gentle mechanical stimulation of non-glandular trichomes by brushing significantly enhances shoot Cd{superscript 2} accumulation in Arabidopsis thaliana, although the underlying physiological mechanism remained unclear. Here, we demonstrate that trichome brushing specifically induces stomatal opening, elevates transpiration, and promotes Cd{superscript 2} uptake, as evidenced by measurements of stomatal aperture, stomatal conductance, transpiration rate, and Cd{superscript 2} concentration in wild-type and mutant leaves. These responses are absent in the glabrous gl1-1 mutant and are not elicited by brushing epidermal pavement cells, indicating a trichome-specific mechanosensory pathway. Mechanistically, this pathway requires MPK3/MPK6 activity and employs malic acid as a key regulator of stomatal aperture, functioning independently of canonical ABA signaling. Our findings uncover a previously unrecognized trichome-to-stomatal signaling cascade that links trichome mechanostimulation to enhanced heavy metal accumulation.

plant biology↗

bioRxiv2025

Allosteric Ligand-Aptamer Complexes Orchestrate Supramolecular or Transient Catalytic, Transcription and Fibrinogenesis Processes

Allosteric regulation, the modulation of biological macromolecular function through binding of molecules at distant sites distinct from the active site, is a fundamental principle in biology that governs enzyme activity, signaling, and gene expression. In this work, we present allosteric ligand/aptamer complexes, coupled to biocatalytic reaction modules composed of enzymes, DNAzymes, or transcription machineries, regulating the catalytic and transient functions of these frameworks. This principle is exemplified by the assembly of ligand/aptamer subunits supramolecular complexes that allosterically stabilize the Mg{superscript 2}-dependent DNAzyme, allowing its ribonucleobase cleavage activity, promoting the formation of transcription templates that yield RNA products, and modulating the assembly of thrombin aptamer subunits that inhibit thrombin-induced coagulation. Specifically, melamine (Mel)/aptamer subunits complexes allosterically stabilize the assembly of Mg{superscript 2}-dependent DNAzyme strands for substrate cleavage, the formation of thrombin aptamer subunits that inhibit the conversion of fibrinogen to fibrin, and the stabilization of a transcription template encoding the Malachite Green (MG) RNA aptamer. Furthermore, coupling an enzyme that depletes the ligand/aptamer complex, which allosterically stabilizes the biocatalytic reaction module, demonstrates the dissipative and transient operation of the catalytic system. This concept is illustrated by the adenosine (Ade)/aptamer subunits supramolecular complex, which stabilizes thrombin aptamer subunits to inhibit thrombin-induced fibrinogenesis, and promotes the formation of an active transcription template for RNA synthesis. In the presence of adenosine deaminase (ADA), Ade is transformed into inosine, which lacks affinity for the aptamer subunits, thereby degrading the Ade/aptamer assemblies and depleting the allosteric complexes. The temporal disassembly of these allosteric stabilizing complexes leads to the transient inhibition of thrombin-induced coagulation or to the transient operation of a transcription machinery. For Table of Contents Only: O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/684577v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1a82199org.highwire.dtl.DTLVardef@135f2d1org.highwire.dtl.DTLVardef@d8b689org.highwire.dtl.DTLVardef@1b7e609_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Complexes between Netrin G Ligands and Chiral Nanoparticles Promote Axons Regeneration under Near-Infrared Illumination

Chiral nanoparticles combine functionalities of inorganic materials and large biomacromolecules enabling stimulation of neurons. However, multiple challenges remain in their use for central nervous system including identifying suitable cell signaling pathways and traversing the blood-brain barrier. In this study, we show that glutathione-coated Ni(OH)2 nanoparticles form nanoscale complexes with netrin-G1 ligands (NGL-1), critical for neuronal regeneration. NGL-1 features a semispherical pocket with a 2.5 nm radius of curvature, fitting well with nanoparticles sized 3 {+/-} 1.2 nm. D-NPs with D-glutathione surface ligands activate D-glutathione receptors on epithelial cells, facilitating their transport into the brain. When illuminated with 980 nm light, the nanoparticle-protein complex stimulates axon regeneration through localized IGF-1 production. This approach successfully regenerated (a) hippocampal neurons in Alzheimers disease mice and (b) dorsal root ganglion neurons in spinal cord-injured mice. The nanoparticles were thoroughly tested for safety and excreted intact. The local, rather than systemic, IGF-1 upregulation minimizes its side effects.

neuroscience↗

bioRxiv2025

DCX Enhances Glioblastoma Metabolism Through Synergistic Regulation of Glutamine Synthesis and Metabolism-Related Genes for Cellular Homeostasis

Gliomas are the most common primary intracranial tumors, comprising 81% of malignant brain tumors, and currently lack effective therapies. Recent advances in molecular biology have shown that cancer cells exploit microtubule-associated proteins (MAPs) under stress to activate various signaling pathways. This study investigates the role of Doublecortin (DCX) in glioma metabolism and its impact on tumor proliferation. In this study, CRISPR-engineered glioma models with DCX overexpression or knockdown were analyzed using integrated genomic, transcriptomic, and metabolomic approaches. Metabolic activity was assessed via RNA sequencing, Seahorse assays, and targeted mass spectrometry. Pharmacological inhibition of key pathways validated functional dependencies. We demonstrate that gliomas enriched with DCX exhibit elevated glycolytic activity while also relying on cellular respiration and oxidative phosphorylation (OXPHOS) for energy to support the abnormal proliferation of glioma cells. Upon integrative analysis of enriched genes and proteins, we observed genetic and metabolome-level signatures associated with differences in central carbon and energy metabolism in CRISPR-modified glioma cells expressing high DCX. Whole-genome transcriptome analysis revealed enriched metabolic entities promoting hydrolysis of glutamine and glutaminolysis in glioma cells and inhibition of selected differentially enriched genes with small molecule inhibitors abrogated metabolic enrichment and resulted in reduced energy levels and protein translation required for aberrant growth. Finally, we establish that DCX stimulates glutaminolysis to regulate homeostasis for energy supplements in glioma cells. Targeting DCX-mediated metabolic pathways may provide a novel therapeutic approach for glioblastoma, highlighting the potential for innovative treatments in this challenging disease.

cancer biology↗

bioRxiv2025

Classification of Urticaceae based on morphology and phylogenetic inference

The Urticaceae (ca. 2600 species) were first formally recognized by Jussieu in the 18th century and last comprehensively monographed by Weddell in the 19th century. Since Weddells work, family delimitation has been modified and many genera described in a fragmented manner. Over the past two decades, numerous molecular studies have supported the inclusion of Cecropiaceae within Urticaceae and identified paraphyly in several genera, notably Laportea, Urera, Boehmeria, Parietaria, Pellionia, and Pouzolzia. However, few studies have translated these molecular insights into a revised taxonomy. This study aimed to provide a robust, updated classification for Urticaceae by: a) increasing taxon and genomic locus sampling through the integration of newly generated sequence data with previously published datasets; and b) incorporating morphological data to support a revised delimitation of tribes and genera, and to establish a new linear sequence for the family. We also sought to identify remaining taxonomic challenges. Using Sanger and Angiosperms353 sequence data, we constructed a phylogenetic framework for 57 out of 59 currently accepted genera. We also assessed the phylogenetic informativeness of 57 morphological characters by mapping them onto the phylogeny. Our analyses support the delimitation of 61 monophyletic genera and an infrafamilial classification comprising seven tribes, two of which we describe as new: Myriocarpeae and Leukosykeae. We provide a revised linear sequence for the family. Our classification reinstates several names previously treated as synonyms (Fleurya, Leptocnide, Margarocarpus, Polychroa, Scepocarpus, Sceptrocnide), places several genera in synonymy (Hemistylus and Rousselia under Pouzolzia; Hesperocnide under Urtica; Gesnouinia and Soleirolia under Parietaria), and proposes the recognition of two new genera, Muimar gen. nov. and Pouzolziella gen. nov., to accommodate Boehmeria nivea and Pouzolzia australis, respectively. Mapping morphological characters onto the phylogeny indicates that while most states are homoplastic at the family level, their combination is valuable for recognizing genera. Geographic character mapping suggests a high degree of spatial conservatism at the genus rank. Our dated ultrametric tree suggests an origin for Urticaceae in Indomalaya during the mid-Cretaceous, followed by establishment in the Laurasian boreotropical flora and subsequent dispersal to the neotropics and Africa. Once classified within an evolutionary framework we believe that the Urticaceae represent a valuable study system in evolutionary biology for investigating transitions across biomes, the drivers of floral trait evolution, and intrinsic speciation mechanisms. New tribes: --Leukosykeae, Myriocarpeae New genera: --Muimar, Pouzolziella

evolutionary biology↗

bioRxiv2025

Precision Editing of Cyclophilin A Generates Cyclosporine and Voclosporin Resistant Cellular Therapies

Recipients of allogeneic transplants or patients with autoimmune disease require immune suppression, often with calcineurin inhibitors. There is an expanding repertoire of immune effector cell therapies, including CD19 CAR-T cells and viral-specific T cells, deployed in these patients; however, ongoing calcineurin inhibition may be detrimental to cell therapy function. We developed a CRISPR/Cas9-based approach to engineer dual cyclosporine/voclosporin resistant cell therapies by targeting PPIA (encoding cyclophilin A), a critical binding partner for both drugs. Because Cyclophilin A has homeostatic functions in T cells, a complete knock-out is detrimental to cell viability. We thus targeted its C-terminus, disrupting drug binding while leaving the majority of the protein intact. C-terminal editing was stable throughout expansion and preserved Cyclophilin A expression. Edited CD19 CAR-T cells retained effector function in the presence of cyclosporine and voclosporin, including proliferation, cytokine production, and target cell killing, resulting in improved survival in murine models of CD19+ leukemia. Edited CMV-specific T cells also demonstrated preserved antigen-specific proliferation and cytokine production in the presence of these drugs. C-terminal editing of Cyclophilin A offers a promising avenue for developing next-generation cell therapies for patients receiving calcineurin inhibitors.

immunology↗

bioRxiv2025

Structural basis of PPARγ-mediated transcriptional repression by the covalent inverse agonist FX-909

Hyperactivation of peroxisome proliferator-activated receptor gamma (PPAR{gamma})-mediated transcription promotes tumor growth in urothelial (bladder) cancer, which can be inhibited by pharmacological compounds that repress PPAR{gamma} activity. FX-909 is a covalent PPAR{gamma} inverse agonist currently in phase 1 clinical trials for advanced solid malignancies including muscle-invasive bladder cancer. Here, we compared the mechanism of action of FX-909 to other covalent inverse agonists including T0070907, originally reported more than 20 years ago and misclassified as an antagonist, and two recently reported improved covalent inverse agonist analogs, SR33068 and BAY-4931. Functional profiling and NMR studies reveal that FX-909 displays improved corepressor-selective inverse agonism and better stabilizes a transcriptionally repressive PPAR{gamma} LBD conformation compared to T0070907. The crystal structure of PPAR{gamma} LBD cobound to FX-909 and NCoR1 corepressor peptide reveals a repressive conformation shared by other covalent inverse agonists. These findings build on recent studies highlighting the pharmacological significance and clinical relevance of transcriptionally repressive PPAR{gamma} inverse agonists.

biophysics↗

bioRxiv2025

RIP2 inhibits DOCK8-mediated platelet dense granule release and alleviates experimental myocardial infarction

ObjectiveReceptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity downstream of pattern recognition receptors (PRRs). Platelets express RIP2, while its role in platelet activation, thrombosis, and myocardial infarction (MI) is unknown. Approach and ResultsHere we show that RIP2 deficiency enhances platelet dense granule secretion in response to GPIb and GPVI activation; platelet aggregation in whole blood and adhesion under arterial shear are also increased. Consistently, RIP2 inhibitor WEHI-345 potentiates human platelet dense granule secretion and inhibits RIP2 phosphorylation induced by thrombin and collagen. These phenotypes are translated into shorter bleeding time, accelerated FeCl3-induced arterial thrombosis. Importantly, platelets from patients with coronary artery disease and mice with atherosclerosis express lower RIP2, and RIP2 deficiency deteriorates MI and cardiac function in a mouse ischemia/reperfusion model. Mechanistically, we found that platelets express dedicator of cytogenesis protein 8 (DOCK8), which is sequestered by phosphorylated RIP2 (p-RIP2), causing inhibition of Cdc42 activation and subsequent dense granule release. ConclusionsIn conclusion, RIP2 inhibits platelet activation, thrombosis, and ameliorates MI. Our results also suggest that a novel PRR-independent pathway, p-RIP2-DOCK8-Cdc42, negatively regulates platelet activation downstream of GPIb and GPVI. Platelet RIP2 pathway may be promising for therapeutic intervention of atherothrombotic diseases from early atherosclerosis stage to arterial thrombosis and MI. HighlightsO_LIRIP2 inhibits platelet dense granule release NOD2-independenly via pRIP2-DOCK8-Cdc42. C_LIO_LIThe negative regulation of RIP2 on platelet activation is translated into enhanced thrombosis, exacerbated myocardial infarction (MI). C_LIO_LIRIP2 pathway booster may be an approach for therapeutic intervention of atherothrombotic diseases. C_LI

molecular biology↗

bioRxiv2025

Identification of chemical features that influence mycomembrane permeation and antitubercular activity

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the deadliest single-agent infection worldwide. Current antibiotic treatment for TB takes a minimum of four months, underscoring the need for better therapeutics. The unique mycobacterial cell envelope, particularly the outermost mycomembrane, has long been thought to promote intrinsic antibiotic resistance by limiting compound entry into Mtb. Understanding chemical features that influence permeation across the mycomembrane may enable more accurate predictions of whole cell anti-Mtb activity, leading to development of more effective antibacterials. Here we query the mycomembrane permeation of over 1500 azide-tagged compounds in live Mtb with the bioorthogonal click chemistry-based assay PAC-MAN. We use cheminformatics and machine learning to identify chemical features associated with mycomembrane permeation and show that they have predictive value via systematic modification of two small molecule series. Additionally, we find that chemical features that influence mycomembrane permeation correlate with anti-Mtb activity in large compound libraries. These findings suggest that the mycomembrane is indeed a significant barrier to whole cell activity in Mtb and provide a rational framework for designing or modifying compounds to overcome this barrier.

microbiology↗

bioRxiv2025

Themis dominates T cell exhaustion by regulation of TCR and PD-1 signaling

T cell exhaustion is important to protect the host from immunopathology during chronic viral infection, but it also impairs T cell anti-tumor immunity1-5. A fundamental unresolved question is whether and how T cell exhaustion is determined at the onset of TCR signaling6-8. Here we report an unexpected role of Themis, a TCR-proximal signaling molecule9, in T cell exhaustion. Chronic viral infection in mice usually leads to T cell exhaustion and survival of the host. Surprisingly, Themis T-cell conditional knockout mice died from severe CD8+-dependent lung immunopathology in chronic viral infection, showing Themis importance in establishing T cell exhaustion. We found that Themis-deficient CD8+ T cells were hyperactivated at the single-cell level - producing more TNF and IFN{gamma} compared to wild-type counterparts - but defective in population-level expansion. Moreover, TCF-1 and TOX expression were inhibited in Themis-deficient CD8+ T cells, thereby impairing differentiation of exhausted T cell precursors (T-pex) and maintenance of terminally exhausted T cells (T-ex), respectively. Mechanistically, Themis initially promotes TCR signaling to induce PD-1 expression and subsequently mediates PD-1 signaling. In the latter, Themis binds to PD-1 and promotes PD-1 phosphorylation and its recruitment of SHP2, thereby acting as a negative regulator to inhibit T cell effector functions. Without Themis, the orderly regulation of TCR and PD-1 signaling, and therefore exhaustion, is disrupted. Thus, our results unequivocally demonstrate that Themis-mediated early TCR signaling plays a decisive role in T cell exhaustion and provide a novel mechanism of PD-1 signaling through Themis.

immunology↗

bioRxiv2025

A dual interaction between RSV NS1 and MED25 ACID domain reshapes antiviral responses

Respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in infants, elicits a remarkably weak innate immune response. This is partly due to the type I interferon (IFN-I) antagonism of the non-structural RSV NS1 protein. It was recently suggested that NS1 could modulate host transcription via an interaction with the MED25 subunit of the Mediator complex. Previous work emphasized the role of the NS1 C-terminal helix 3 for recruitment of the MED25 ACID domain, a target of transcription factors (TFs). Here we show that the NS1 /{beta} core domain binds to MED25 ACID, and acts cooperatively with NS1 3 to achieve nanomolar affinity. This strong interaction is rationalized by the dual NS1 binding site on MED25 ACID predicted by AlphaFold3, which overlaps with the two canonical binding interfaces of TF transactivation domains (TADs), H1 and H2. By NMR, we confirmed that the NS1 /{beta} core domain targets the H2 interface. Several single amino acid NS1 /{beta} core domain mutations displayed reduced affinity for MED25 ACID, both in vitro and in cellula, at a comparable extent to the deletion of NS1 3. These mutations resulted in attenuated replication of recombinant RSV (rRSV) and increased expression of several antiviral interferon-stimulated genes (ISG) in interferon-competent cells. In MED25 knockdown cells, rRSV-mCherry replication was further attenuated, in line with the upregulation of IFT1 and ISG15 ISGs. The difference between WT and NS1 mutant rRSV-mCherry was partially lost, suggesting that RSV uses MED25 to control antiviral responses, by a mechanism involving the NS1-MED25 ACID complex. The strong interaction and the extended binding surface of NS1 on MED25 ACID provide evidence for a mechanism, where NS1 blocks access of transcription factors to MED25 ACID, and thereby MED25 mediated transcription activation. Author SummaryRespiratory syncytial virus (RSV) is a major pathogen for acute lower respiratory infections in infants and in the elderly. RSV elicits a remarkably weak immune response. It has developed a unique strategy to counteract the immune system, by encoding two small multifunctional proteins, RSV NS1 and NS2. NS1 is involved in interferon antagonism in the cytosol. Recently NS1 was shown to modulate host transcription in the nucleus. However, the mechanisms underpinning this function are not fully clear. Here we focus on the interplay between NS1 and the cellular MED25 coactivator protein, which can contribute to the antiviral response by activating several innate immune response genes. The MED25 C-terminal ACtivator Interacting Domain (ACID), a target of cellular transcription factors (TF), is a key feature for this function. To investigate the impact of MED25 hijacking by NS1, we combined in vitro biophysical experiments and cellular assays to probe the relationship between the stability of the NS1-MED25 ACID complex and RSV replication as well as antiviral responses. Our results suggest that this interaction is correlated with antiviral response antagonism, probably by hindering TFs to interact with MED25-ACID. This knowledge might pave the way for antiviral strategies aimed at stimulating appropriate immune responses.

pathology↗

bioRxiv2025

WormID-Benchmark: Extracting Whole-Brain Neural Dynamics of C. elegans At the Neuron Resolution

The nematode C. elegans is a premier model organism for studying neural circuit function due to its fully mapped connectome and genetically identifiable neurons. Recent advances in 3D light microscopy and fluorescent protein tagging have enabled whole-brain imaging at single-neuron resolution. However, extracting meaningful neural dynamics from these high-resolution recordings requires addressing three fundamental challenges: (i) accurate detection of individual neurons in fluorescence images, (ii) precise identification of neuron classes based on anatomical and colorimetric cues, and (iii) robust tracking of neurons over time in calcium imaging videos. To systematically evaluate these challenges, we introduce WormID-Bench, a large-scale, multi-laboratory dataset comprising 118 worms from five distinct research groups, along with standardized evaluation metrics for detection, identification, and tracking. Our benchmark reveals that existing computational approaches show substantial room for improvement in sensitivity, specificity, and generalization across diverse experimental conditions. By providing an open and reproducible benchmarking framework1, WormID-Bench aims to accelerate the development of high-throughput and scalable computational tools for whole-brain neural dynamics extraction in C. elegans, setting the stage for broader advancements in functional connectomics.

neuroscience↗

bioRxiv2024

scCompass: An integrated cross-species scRNA-seq database for AI-ready

Emerging single-cell sequencing technology has generated large amounts of data, allowing analysis of cellular dynamics and gene regulation at the single-cell resolution. Advances in artificial intelligence enhance life sciences research by delivering critical insights and optimizing data analysis processes. However, inconsistent data processing quality and standards remain to be a major challenge. Here we propose scCompass, which provides a data quality solution to build a large-scale, cross-species and model-friendly single-cell data collection. By applying standardized data pre-processing, scCompass integrates and curates transcriptomic data from 13 species and nearly 105 million single cells. Using this extensive dataset, we are able to archieve stable expression genes (SEGs) and organ-specific expression genes (OSGs) in human and mouse. We provide different scalable datasets that can be easily adapted for AI model training and the pretrained checkpoints with state-of-the-art (SOTA) single-cell foundataion models. In summary, the AI-readiness of scCompass, which combined with user-friendly data sharing, visualization and online analysis, greatly simplifies data access and exploitation for researchers in single cell biology(http://www.bdbe.cn/kun).

bioinformatics↗

bioRxiv2024

Spatiotemporal transcriptomic analyses reveal molecular gradient patterning during development and the tonotopic organization along cochlear axis

While cochlear tonotopic organization is essential for sound frequency discrimination, the spatiotemporal molecular programs shaping this architecture remain unresolved. Here, we integrate spatiotemporal transcriptomics (E17.5-P8-adult) with single-cell RNA-seq to construct a single-cell-resolution atlas of mouse cochlear development. As marked by differential gradient expression genes, distinct spatially and molecularly defined hair cell (HC) subtypes along the apical-basal axis were identified: such as the apical Myo7a+++/Calb1+++ outer hair cell (OHC) subtype, the basal Myo7a+/Calb1{square} OHC subtype, the apical vGlut3+++/Calb2+ inner hair cell (IHC) subtype, and the basal vGlut3+/Calb2+++ IHC subtype. In addition, the spatial data also demonstrate the developmental reversal of Myo7a/Calb2Nefh gradient patterns in HCs and spiral ganglion neurons (SGNs), revealing dynamic plasticity and complexity in frequency coding during development. Spatial analyses further demonstrate regional heterogeneity in cell communication intensity between HCs and SGNs, and also reveal core signaling hubs coordinating tonotopic organization, such as Fgf10-Fgfr2 pathway in the basal region and Ptn-Sdc pathway in the apical region HC or SGNs at P8 stage. Our study provides an open-access spatial database and reveals the morphological and molecular foundations underlying cochlear tonotopic organization, linking molecular and developmental mechanisms to auditory pathophysiology.

developmental biology↗

bioRxiv2024

Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2, and Triobp genes

The RFX family of transcription factors plays crucial roles in the regulation of ciliogenesis and organ development. Mutations of RFX transcription factors lead to various genetic diseases, including ciliopathies and hearing loss, although the underlying mechanisms remain unclear. This study comprehensively analyzed the expression patterns of RFX family members during different inner ear developmental stages. Rfx3 and Rfx7 were identified as the dominantly expressed members in cochlear hair cells, exhibiting alternative splicing variants and dynamic subcellular localization at different stages. Integration of single-cell transcriptomics, ChIP-seq, and ATAC-seq data indicates that Rfx3 functions as either a transcriptional activator or repressor, binding to numerous genes related to ciliogenesis, hair bundle structures, and planar cell polarity. Rfx3 regulates the spatiotemporal expression of hair bundle gene Triobp in hair cells by binding to and modulating its intronic enhancer. Additionally, Rfx3 binds to hair cell differentiation and fate determination genes Tbx2, Insm1, and Ikzf2. The results demonstrate that Rfx3 and Rfx7 are dominantly expressed in outer hair cells, with their subcellular localization shifting to cytoplasm at later developmental stages. This suggests a previously unknown function of Rfx3 and Rfx7 beyond transcriptional regulation, highlighting their complex roles in hair cell differentiation and maintenance.

developmental biology↗

bioRxiv2024

Peptide-MHC-targeted retroviruses enable in vivo expansion and gene delivery to tumor-specific T cells

Tumor-infiltrating-lymphocyte (TIL) therapy has demonstrated that endogenous T cells can be harnessed to initiate an effective anti-tumor response. Despite clinical promise, current TIL production protocols involve weeks-long ex vivo expansions which can affect treatment efficacy. Therefore, additional tools are needed to engineer endogenous tumor-specific T cells to have increased potency while mitigating challenges of manufacturing. Here, we present a strategy for pseudotyping retroviral vectors with peptide-major histocompatibility complexes (pMHC) for antigen-specific gene delivery to CD8 T cells and examine the efficacy of these transduced cells in immunocompetent mouse models. We demonstrate that pMHC-targeted viruses are able to specifically deliver function-enhancing cargoes while simultaneously activating and expanding anti-tumor T cells. The specificity of these viral vectors enables in vivo engineering of tumor-specific T cells, circumventing ex vivo manufacturing processes and improving overall survival in B16F10-bearing mice. Altogether, we have established that pMHC-targeted viruses are efficient vectors for reprogramming and expanding tumor-specific populations of T cells directly in vivo, with the potential to substantially streamline engineered cell therapy production for a variety of applications.

immunology↗

bioRxiv2024

Advancing Variant Phenotyping in Myelodysplastic Syndromes via Computational Genomics of Mitochondrial Enzyme Complexes

Mitochondria are essential cellular organelles that play critical roles in hematological disorders. Recurrent mutations in mitochondrial DNA (mtDNA) have been identified in patients with myelodysplastic syndromes (MDS) and serve as significant prognostic indicators for their outcomes following allogeneic hematopoietic stem-cell transplantation (allo-HCT). However, the biological mechanisms of mtDNA mutations remain unclear. The current study utilizes computational structural genomics to improve our understanding of pathogenic variants in mitochondria-encoded genes. This emerging genomics discipline employs structural models, molecular mechanic calculations, and accelerated molecular dynamic simulations to analyze gene products, focusing on their structures and motions that determine their function. We applied this methodology to perform deep variant phenotyping of entire mitochondria-encoded protein complexes associated with the pathobiology of MDS and their prognosis after HCT. Our results demonstrate that this approach significantly outperforms conventional analytical methods, providing enhanced and more accurate information to support the potential pathogenicity of these variants and better infer their dysfunctional mechanisms. We conclude that the adoption and further expansion of computational structural genomics approaches, as applied to the mitochondrial genome, have the potential to significantly increase our understanding of molecular mechanisms underlying the disease. Our study lays a foundation for translating mitochondrial biology into clinical applications, which is of significant mechanistic and biomedical relevance and should be considered in modern biomedical research.

genomics↗