Biology subjects

He, M.

Publications and source records attributed to He, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Albumin-STING Nanocomplex Reprograms HSPCs to Antitumor Neutrophils with Enhanced MHC I Antigen Presentation for Cancer Immunotherapy

Tumor-associated immunosuppressive neutrophils, termed myeloid-derived suppressor cells (MDSCs), compromise cancer immunotherapy. They can be pathologically programmed as early the hematopoietic stem and progenitor cell (HSPC) stage by suppressing interferon signaling. Reprogramming HSPCs toward antitumor neutrophils through the stimulator of interferon genes (STING) activation offers a promising therapeutic strategy. Here, we demonstrate that an albumin-STING nanoagonist (Nano ZSA-51D) reprograms HSPCs to generate antitumor neutrophils, enhancing MHC I-mediated CD8 T cell immunity. Nano ZSA-51D activates STING-interferon signaling in HSPCs, promoting their expansion and differentiation toward granulocyte-monocyte progenitors via STING-NF-{kappa}B-IL-6 signaling. It further reprograms neutrophils into CD14ICAM-1+ subset through STING-NF-{kappa}B-TNF- signaling, enhancing tumor infiltration. These neutrophils upregulate interferon signaling and MHC I antigen presentation, boosting tumor-specific CD8 T cell responses. Adoptive transfer of Nano ZSA-51D-reprogrammed neutrophils with -PD1 therapy achieves complete colon tumor remission. Our findings provide a novel strategy to reprogram HSPCs toward antitumor neutrophils and highlight the potential of early interventions at HSPC stage to rewire neutrophil fate for cancer immunotherapy.

immunology↗

bioRxiv2025

Deficiency in POLE exonuclease causes synthetic lethality in highly aneuploid cancer cells

Aneuploidy is a hallmark of cancer and is associated with drug resistance and poor clinical outcomes across diverse cancer types. However, no therapies have been clinically established to target highly aneuploid tumors. By analyzing a real-world dataset comprising nearly half a million tumor samples subjected to comprehensive genomic profiling, we identified a striking mutual exclusivity between POLE exonuclease domain mutations and high aneuploidy burden. This observation was independently validated using data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). Probabilistic modeling revealed that the elevated quantity and unique spectrum of mutations induced by POLE exonuclease deficiency increase the likelihood of inactivating essential genes on chromosome arms harboring losses, leading to a synthetic lethal phenotype in highly aneuploid cells. Functional experiments demonstrated that POLE exonuclease activity is essential for the viability of highly aneuploid cancer cell lines but is dispensable in diploid cells. These findings suggest that selective inhibition of POLE exonuclease may represent a promising therapeutic strategy for targeting highly aneuploid tumors.

cancer biology↗

bioRxiv2025

Characterization and Genome Insights of Two Novel Plastic-Degrading Acinetobacter Species

The ubiquitous presence of Acinetobacter species in the environment holds significant biotechnology potential, particularly in the degradation of various pollutants. In this study, we present two plastic-degrading bacteria, strains CAAS 2-6T and CAAS 2-13T, isolated from landfill leachate and a strawberry farmland, respectively. Strain CAAS 2-6T showed the highest 16S rRNA sequence similarity (97.7%) with Acinetobacter gerneri DSM 14967T, while strain CAAS 2-13T was most closely related (99.6%) to Acinetobacter gerneri PS-1T. Phylogenetic analysis of 16S rRNA and gyrB-rpoB genes placed both strains on distinct branches. Genomic comparisons revealed the highest digital DNA-DNA hybridization (dDDH) / average nucleotide identity (ANI) values for CAAS 2-6T with Acinetobacter indicus CIP 110367T (22.7%/77.0%) and for CAAS 2-13T with Acinetobacter gerneri PS-1T (66.5%/96.0%). Based on phenotypic and chemotaxonomic data, we propose strain CAAS 2-6T as the novel species Acinetobacter lentus sp. nov. (type strain CAAS 2-6T = GDMCC 1.3951T = JCM 36321T = KCTC 8156T), and strain CAAS 2-13T as the novel subspecies Acinetobacter kanungonis subsp. fragariae subsp. nov. (type strain CAAS 2-13T = GDMCC 1.3956T = JCM 36322T = KCTC 8157T). Both strains utilized PLA, PBSA, PBS, PBAT, and PBT as sole carbon sources, with CAAS 2-6T exhibiting superior growth on PBSA, PBS, and PBAT. Genomic annotation identified genes encoding plastic-degrading enzymes multicopper oxidase AbMCO and alkane hydroxylase AlkB. Enzymatic depolymerization assays confirmed in vitro production of plastic monomers. These findings expand the known metabolic capabilities of Acinetobacter and provide promising new candidates for plastic bioremediation. IMPORTANCEAcinetobacter spp. demonstrate significant potential for plastic bioremediation. We characterize two novel strains: CAAS 2-6T (A. lentus sp. nov.) and CAAS 2-13T (A. kanungonis subsp. fragariae subsp. nov.), capable of degrading five major polyesters (PLA, PBAT, PBT, PBS, PBSA) as sole carbon sources. Genomic and enzymatic analyses identified multicopper oxidase (AbMCO) and alkane hydroxylase (AlkB) as key depolymerase. This work: (i) Expands plastic-degrading Acinetobacter diversity; (ii) Provides novel biocatalysts and genomic resources; (iii) Delivers engineerable microbial chassis.

microbiology↗

bioRxiv2025

Neurogranin Unlocks the L-type Ca2+ Channel and Directs Calmodulin Delivery

Neurogranin (Ng) is enriched in the postsynaptic density, with mounting evidence of its importance to neural pathophysiology. Ng has long been recognized as a dedicated calmodulin (CaM)- binding/buffering protein. This study demonstrates that Ng, through its CaM-binding IQ domain, directly binds the distal C-terminus (DCT) of the L-type Ca2+ (CaV1) channel, as a de novo mechanism regulating excitation-transcription coupling. The Ng/DCT interaction attenuates autonomous C- terminus-mediated inhibition and promotes specific CaM delivery to CaV1, cooperatively enhancing channel gating (activation and inactivation), Ca2+ influx and downstream neuronal signaling. Phosphorylation of Ng leads to the dissociation of the CaM C-lobe, while transiently forming the linkage with the CaM N-lobe. In contrast to neurodegenerative disease-related Ng mutants, WT Ng, within the above transient complex, enables the "targeted CaM transfer" to the proximal C-terminus of CaV1. Collective evidence from electrophysiology, binding assays, structural modeling and Ca2+ imaging supports a paradigm of dynamic intra- and inter-molecular interactions/facilitations underlying Ng and CaV1, both of which are representative CaM-binding proteins. This work proposes a mechanistic scheme potentially applicable to a broad scope of CaM-related physiology.

biophysics↗

bioRxiv2025

Cryo-EM structure of DNA-unbound human MCM2-7 complex reveals new disease-relevant regulation

Chromatin loading of the hexameric replicative helicase MCM2-7 complex requires coordinated interactions with the origin recognition complex (ORC), CDC6, and CDT1. MCM2-7 not bound to DNA forms a single hexamer (SH) with an open DNA entry gate. Two MCM2-7 SHs are loaded sequentially to form the double hexamer (DH) that encircles the DNA duplex. Activated MCM2-7 then unwinds DNA and initiates DNA replication. Our cryo-electron microscopy analyses show that a fraction of human MCM2-7 without DNA exists as DH. Unexpectedly, we find that the MCM3 winged helix domain (WHD) docks on MCM2 in both DNA-free DH and SH, creating a safety latch across the DNA entry gate to block DNA entry into the central channel. The safety latch can be opened by ORC-CDC6 binding. Disrupting this latch by designed or human disease-related mutations of MCM3 causes replication defects and DNA damage checkpoint activation. Our findings uncover a new regulated step in MCM2-7 loading with implications for human diseases.

biophysics↗

bioRxiv2025

Novel mouse model reveals neurodevelopmental origin of PMM2-CDG brain pathology

Congenital disorders of glycosylation (CDG) are a group of neurogenetic conditions resulting from disruptions in the cellular glycosylation machinery. The majority of CDG patients have compound heterozygous pathogenic variants in the phosphomannomutase 2 (PMM2) gene. Individuals with PMM2-CDG exhibit multi-systemic symptoms, prominently featuring neurological deficits with nearly all patients exhibiting cerebellar hypoplasia and ataxia. To overcome embryonic lethality caused by whole body knock-out of Pmm2 and mimic patient-related compound heterozygous pathogenic variants, we paired a Pmm2 flox allele (Pmm2fl) with a catalytically inactive knock-in allele (Pmm2R137H), commonly present in PMM2-CDG patients. Mice with post-mitotic loss of PMM2 from neurons or astrocytes are indistinguishable from unaffected littermates, including in a broad battery of neurological assessments. In contrast, removal of PMM2 from embryonic neural precursor cells leads to cerebellar hypoplasia, ataxia, seizures, and early lethality. Comprehensive multi-omics profiling, including metabolomics, glycomics, single-cell transcriptomics, proteomics, and glycoproteomics, reveal widespread molecular disturbances throughout the brain, with the cerebellum showing the most pronounced disruption. These findings highlight the heightened dependency of the developing cerebellum on intact N-glycosylation, aligning with clinical observations in PMM2-CDG patients. Importantly, glycoproteomic alterations identified in our mouse model are corroborated in PMM2-CDG patient post-mortem cerebellar tissue, underscoring the translational relevance of our findings and implicating impaired synaptic transmission as a key pathogenic mechanism.

neuroscience↗

bioRxiv2025

eyeris: A flexible, extensible, and reproducible pupillometry preprocessing framework in R

Pupillometry provides a non-invasive window into the mind and brain, particularly as a psychophysiological readout of autonomic and cognitive processes like arousal, attention, stress, and emotional states. Pupillometry research lacks a robust, standardized framework for data preprocessing, whereas in functional magnetic resonance imaging and electroencephalography, researchers have converged on tools such as fMRIPrep, EEGLAB and MNE-Python. Many established pupillometry preprocessing packages and workflows fall short of serving the goal of enhancing reproducibility, especially since most existing solutions lack designs based on Findability, Accessibility, Interoperability, and Reusability (FAIR) principles. To promote FAIR and open science practices for pupillometry research, we developed eyeris, a complete pupillometry preprocessing suite designed to be intuitive, modular, performant, and extensible (https://github.com/shawntz/eyeris). Out-of-the-box, eyeris provides a recommended preprocessing workflow and considers signal processing best practices for tonic and phasic pupillometry. Moreover, eyeris further enables open and reproducible science workflows, as well as quality control workflows by following a well-established file management schema and generating interactive output reports for both record keeping/sharing and quality assurance of preprocessed pupil data prior to formal analysis. Taken together, eyeris provides a robust all-in-one transparent and adaptive solution for high-fidelity pupillometry preprocessing with the aim of further improving reproducibility in pupillometry research.

neuroscience↗

bioRxiv2025

Azapeptide-based GLP-1 receptor agonist improves glucose metabolism in a diet-induced obesity model

Peptide therapeutics are limited by rapid proteolysis and short half-lives. Azapeptides, created by replacing one or more -carbon(s) on the peptide backbone with nitrogen atom(s), offer a strategy to improve peptide stability while preserving functional efficiency, yet their clinical potential has remained underexplored. Herein, we report the design, synthesis, in vitro and in vivo evaluations of azapeptide-based glucagon-like peptide-1 receptor agonists (GLP-1RAs). Using a solid-phase synthesis platform, we generated GLP-1 analogues with aza-substitutions at protease-sensitive residues. The lead analogue, AzaA8/R34-GLP-1(AzaA8), resisted dipeptidyl peptidase-4 degradation (>24 h), maintained picomolar potency at the GLP-1 receptor (GLP-1R) signaling, and exhibited an extended plasma half-life in mice relative to unmodified controls. In lean mice, AzaA8 improved oral glucose tolerance, and in high-fat diet-induced obese mice, chronic administration reduced body weight, decreased leptin and insulin levels, and enhanced glucose handling without detectable inflammatory adverse effects. These findings demonstrate that a targeted aza-substitution yields a protease-stable, biologically active GLP-1RA with metabolic benefits, establishing azapeptides as a promising scaffold for next-generation incretin-based therapies in diabetes and obesity.

biochemistry↗

bioRxiv2025

Unraveling Tyrosine-Kinase Inhibitor Resistance in NSCLC Cells via Same-cell Measurement of RNA, Protein, and Morphological Responses

Non-small cell lung cancer (NSCLC) frequently develops resistance to tyrosine kinase inhibitors (TKIs), limiting the long-term success of targeted therapies. A deeper understanding of resistance mechanisms at the molecular and cellular levels may enable the development of more effective treatment strategies. Here, we applied the Teton detection assay on the AVITI24 platform to measure concurrently RNA, protein, and cellular morphology in NSCLC cell lines treated with the TKIs gefitinib and osimertinib. This single-cell, multiomic analysis revealed distinct expression and morphological profiles between drug-sensitive and resistant cells, including differences in MAPK-related pathway activity. Stratifying responses at the single-cell level uncovered subtle responses not detectable in bulk measurements. We identified CDK4/6 activity as a route of cell survival under TKI treatment and demonstrated that co-treatment with the CDK4/6 inhibitor palbociclib enhanced TKI efficacy. The ability to measure multiomics and cellular morphology in the same cells opens new avenues for future studies aimed at improving personalized treatment strategies in NSCLC and overcoming the obstacles posed by drug resistance.

genomics↗

bioRxiv2025

AID facilitates TET2 demethylation of Irf4 for plasma cell fate in germinal center B cells

Activation-induced cytidine deaminase (AID) is essential for B cell affinity maturation. We investigated why AID deficiency gives rise to giant germinal centers (GC) using the AIDR112H mouse model that is devoid of AID activity. The increased GC response was associated with accumulation of GC B cells in the light zone in immunized AIDR112H mice. AIDR112H GC B cells had reduced capacity to up-regulate IRF4 to initiate plasma cell differentiation, leading to accumulation of a transitional GC population with reduced GL7 expression. Genetic introduction of a high affinity B cell receptor (BCR) was unable to restore plasma cell differentiation of AIDR112H B cells while ectopic expression of catalytic active AID rescued plasma cell generation. AIDR112H impaired recruitment of AID to the Irf4 promoter/enhancer and disrupted the interaction with Ten-eleven Translocation 2 (TET2). Consequently, DNA demethylation at the Irf4 promoter/enhancer was reduced in AIDR112H GC B cells and impeded high Irf4 expression for transition into plasma cells. This data reveals a B cell-intrinsic mechanism that governs the plasma cell fate decision through epigenetic remodeling mediated by AID in cooperation with TET2. Key messagesO_LIAID deficiency leads to accumulation of a transitional (t)GC population of B cells with low expression of GL7 and failure to up-regulate IRF4 C_LIO_LIAID deamination activity is needed to promote up-regulation of IRF4 for plasma cell differentiation. C_LIO_LICo-operation between TET2 and AID facilitates demethylation of the Irf4 enhancer/promoter. C_LI

immunology↗

bioRxiv2025

TestNet: A Testing Method for Inferring Microbial Networks with False Discovery Rate Control, for Clustered and Unclustered Samples

Most existing methods for inferring microbial networks generate only point estimates of Pearsons correlations without assessing their significance, and none can account for clustered samples. In this article, we introduce TestNet, a novel method that delivers well calibrated results by controlling the false discovery rate (FDR). We developed a permutation-based procedure to generate valid null replicates that account for compositional effects and extensive zeros in microbiome data, and clustering structures within the samples when present. Our results demonstrate that TestNet is the only method that effectively controls the FDR while maintaining high power across a wide range of scenarios.

bioinformatics↗

bioRxiv2025

Fast in vivo deep-tissue 3D imaging with selective-illumination NIR-II light-field microscopy and aberration-corrected implicit neural representation

Near-infrared II (NIR-II) microscopy, which enables in vivo deep-tissue visualization of vasculature and cell activities, has been a promising tool for understanding physiological mechanisms. However, the volumetric image speed of the NIR-II microscopy is hindered by scanning strategy, causing limitations for observing instantaneous biological dynamics in 3D space. Here, we developed a NIR-II light-field microscopy (LFM) based on selective-illumination and self-supervised implicit neural representation (INR)-reconstruction, which allows ultra-fast 3D in vivo imaging (100 volumes/s). Through integrating INR with view-wise aberration correction, we could conquer the artifacts induced by the angular subsampling and refractive index variation, achieving single-cell resolution at a volume of 550 m diameter and 200 m thickness. The volumetric selective-illumination overcomes the influence of out-of-focus background, together with the low scattering advantage of NIR-II wavelength, extending the imaging depth to 600 m. The developed aberration-corrected implicit neural representation reconstruction (AIR) NIR-II LFM showcases its capability by monitoring hemodynamics of mouse brain under norepinephrine and flow redistribution of ischemic stroke in 3D vasoganglion, as well as noninvasively tracking immune cell activities inside subcutaneous solid tumor through intact skin. This approach represents a significant advancement in 3D in vivo imaging, holding great potential in biomedical research and preclinical studies.

neuroscience↗

bioRxiv2025

RPGR regulates motile cilia by interfering with actin dynamics

Cilia are highly conserved cellular organelles extruding from the surface of cell types carrying either sensory (signaling) or motile functions. These include photoreceptor cells and airway epithelia, where they function in light sensation and mucociliary clearance respectively. Retinitis pigmentosa GTPase regulator (RPGR) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and in some cases, primary ciliary dyskinesia (PCD), both debilitating conditions. Not all patients develop PCD and it is unclear which RPGR variants predispose patients to PCD and why this happens. In this study, using nasal biopsy samples of patients with RPGR-related RP, we leverage 2D organoid cell culturing, super-resolution microscopy, and live cell imaging to characterize the multiciliated cells from patients with different RPGR variants, healthy human nasal and bronchial multiciliated cells with CRISPR-modified RPGR function. We demonstrate for the first time that multiciliated cells with RPGR variants may have reduced ciliation, shorter cilia, significantly impaired cilia beat, or cilia beat incoordination, which could lead to defective mucociliary clearance and lung disease. In addition, we show the regulation of motile cilia by RPGR involves F-actin, as evidenced by temporarily reduced Gelsolin and undissolved condensed actin meshwork at the apical surface of RPGR-deficient multiciliated cells. In support, we show that the motile cilia defect can be ameliorated by treating with the actin polymerization inhibitor Latrunculin A. Though PCD was observed only in patients with variants that affect both main isoforms (RPGR1-19 and RPGRORF15), patients with variants affecting only RPGRORF15 also showed cilia and airway anomalies. Though all RPGR variants affected motile cilia in one way or another, RPGR loss of function variants affecting both isoforms are associated with more severe cilia and systemic phenotypes, the mechanisms of which involve the accumulation of apical F-actin. One Sentence Summary: Loss of RPGR, through the mechanism of apical F-actin accumulation in airway multiciliated cells, leads to reduced ciliation with short cilia that have an impaired beat, leading to defective mucociliary clearance.

cell biology↗

bioRxiv2025

A tumor necrosis factor-α responsive cryptic promoter drives overexpression of the human endogenous retrovirus ERVK-7

Endogenous retroviruses (ERVs) shape human genome functionality and influence disease pathogenesis, including cancer. ERVK-7, a significant ERV, acts as an immune modulator and prognostic marker in lung adenocarcinoma (LUAD). Although ERVK-7 overexpression has been linked to the amplification of the 1q22 locus in approximately 10% of LUAD cases, it predominantly arises from alternative regulatory mechanisms. Our findings indicate that the canonical 5 long terminal repeat (LTR) of ERVK-7 is methylated and inactive, necessitating the use of alternative upstream promoters. We identified two novel transcripts, ERVK-7.long and ERVK-7.short, arising from distinct promoters located 2.8 kb and 13.8 kb upstream of the 5LTR of ERVK-7, respectively. ERVK-7.long is predominantly overexpressed in LUAD. Through comprehensive epigenetic mapping and single-cell transcriptomics, we demonstrate that ERVK-7.long activation is predetermined by cell lineage, specifically in small airway epithelial cells (SAECs), where its promoter displays tumor-specific H3K4me3 modifications. Single-cell RNA sequencing further reveals a distinct enrichment of ERVK-7.long in LUAD tumor cells and alveolar type 2 epithelial cells, underscoring a cell-type-specific origin. Additionally, inflammatory signaling significantly influences ERVK-7 expression; TNF- enhances ERVK-7.long, while interferon signaling preferentially augments ERVK-7.short by differential recruitment of NF-{kappa}B/RELA and IRF to their respective promoters. This differential regulation clarifies the elevated ERVK-7 expression in LUAD compared to lung squamous cell carcinoma (LUSC). Our study elucidates the complex regulatory mechanisms governing ERVK-7 in LUAD and proposes these transcripts as potential biomarkers and therapeutic targets, offering new avenues to improve patient outcomes.

cancer biology↗

bioRxiv2025

Azapeptide Synthesis Fully Integrated into Standard Solid-Phase Peptide Synthesis (SPPS): Case Study with Azapeptide-GLP-1

Azapeptide modification, achieved by substituting backbone -carbons with nitrogen atoms to form enzyme-resistant semicarbazide bonds, can markedly enhance peptide stability and therapeutic potential. However, broad application has been constrained by two major synthetic challenges: the lack of suitable building blocks and the reduced nucleophilicity of the semicarbazide amino group, which limits post-coupling efficiency in automated solid-phase peptide synthesis (SPPS). Here, we describe a fully automated azapeptide synthesis platform that employs Fmoc-protected benzotriazole esters as bench-stable, pre-activated aza-amino acid building blocks. Microwave-assisted synthesis was integrated to accelerate aza-residue incorporation and improve coupling efficiency. This plug-and-play approach enables rapid solid-phase assembly of azapeptides, substantially reducing reaction times and improving yields. To demonstrate its utility, we synthesized azapeptide analogues of glucagon-like peptide-1 (GLP-1) with targeted substitutions at protease-sensitive sites, achieving enhanced stability as demonstrated in our recent study (bioRxiv 2025). This automated platform overcomes long-standing barriers in azapeptide chemistry, providing a scalable route for the rapid generation of stabilized peptide therapeutics. Significance StatementAzapeptides are a promising class of peptidomimetics with enhanced enzymatic stability and therapeutic potential. Yet, their synthesis has been limited by two persistent barriers: the lack of suitable, stable building blocks and the reduced coupling efficiency caused by the semicarbazide backbone. We developed a fully automated, microwave-assisted solid-phase synthesis platform that overcomes both challenges by using bench-stable benzotriazole ester aza-amino acid building blocks. This plug-and-play system enables efficient incorporation of aza-residues under standard SPPS conditions, reducing reaction times while improving yields and reproducibility. The resulting platform allows rapid generation of azapeptide libraries for biological screening and drug development, representing a major step toward scalable production of protease-resistant peptide therapeutics.

biochemistry↗

bioRxiv2025

Ion Channel Reaction Networks: Dielectric Screening and the Importance of Off-Pathway Flux

The transport of ions through channels involves multiple rare-event transitions through a web of interconnected intermediates. Extracting open channel mechanisms generally requires quantifying the relative flux through these intermediates in response to a range of electrochemical gradients. Although this is ideally suited to network-based representations like Markov state models (MSMs), the relative contributions from different pathways and the importance of network resolution remain open areas of research. Herein, we use a complementary approach called multiscale responsive kinetic modeling (MsRKM) to explore how the screening of ionic interactions and the competition between multiple mechanistic pathways contribute to channel mechanisms and current profiles of ion channels. We find that explicitly optimizing screened ionic interactions in the MsRKM framework vastly reduces the solution search space, enabling more efficient identification of physically robust solutions. Using a model of the Shaker Kv channel, we demonstrate that even when systems are well described by a single dominant flux pathway, the remaining contributing pathways and off-pathway flux play multiple essential roles, including shifting current profiles and mechanisms in response to different electrochemical gradients. We additionally discover that current continues to change above the experimentally predicted saturation point. Model systems explain how the degree of dielectric screening influences channel occupancy, the number of contributing pathways, and why current increases or decreases above its experimental saturation point. Our findings emphasize the importance of retaining a full network description to identify and understand ion channel mechanisms. Toc O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/637869v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1144eaaorg.highwire.dtl.DTLVardef@387f8eorg.highwire.dtl.DTLVardef@13e4e01org.highwire.dtl.DTLVardef@1732864_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

A cytoskeletal scaffold promotes motile cilia assembly by regulating transition-zone integrity

Motile cilia are eukaryotic organelles with essential chemo- and mechano-sensing functions across evolution, from single cell organisms to humans. Motile cilia of the mammalian nervous, respiratory and reproductive systems are characterized by unique motility proteins to generate fluid flow essential for transporting metabolites and removing mucus. The molecular mechanism of motile cilia biogenesis remains unknown. Here, we use mouse genetics, single-molecule motility assays, proteomics, high-resolution imaging, and in situ cryo-tomography to identify mammalian KIF27, a motor protein of the Kinesin-4 family and homologue of the Hedgehog pathway regulator COS2/KIF7, as a key regulator of motile cilia assembly. We show that KIF27 promotes the integrity of the transition zone, a diffusion barrier situated at the cilium base. Loss of KIF27 causes specific and profound defects in axonemal structure and disrupts cilia beating, which collectively lead to organismal phenotypes that recapitulate primary ciliary dyskinesia. We show that the motile properties of KIF27 are dispensable for its function in motile cilia biogenesis. Instead, KIF27 acts as a microtubule scaffold to regulate the transition zone architecture and enable correct ciliary incorporation of motility-generating proteins. Given that KIF27 homologues exist in different evolutionarily lineages, we propose that the ancestral activities of KIF27/KIF7 kinesins were to form a microtubule-associated scaffold for protein-protein interactions pertinent to cilia formation and signaling. The transition-zone associated KIF27 activities may represent a general building principle for motile cilia assembly in diverse species and cell types.

developmental biology↗

bioRxiv2024

RNAi-mediated HBV antigen shutdown enhances the antiviral immune effects of PEGIFNalpha via altering T and B cell crosstalk

PEGylated interferon- (PEGIFN) demonstrates promising therapeutic outcomes against chronic hepatitis B (CHB), whereas patient response to PEGIFN therapy remains unsatisfied. Shutdown of hepatitis B virus (HBV) antigens by RNA interference (RNAi) could enhance PEGIFN efficacy in CHB patients, whereas the underlying immunological mechanisms remain obscure. We performed studies by utilizing our newly established extracellular humanized IFNAR (IFNAR-hEC) mice. An in-house constructed small interfering RNAs (GalNac-siHBV) was administrated to mice either alone or in combination with PEGIFN. The phenotypic and functional characteristics of peripheral and organ-specific immune cells were assessed by flow cytometry, ELISpot, RNA sequencing (RNA-seq), and single-cell RNA-seq (scRNA-seq) analysis. Our results demonstrated that combined treatment with PEGIFN and RNAi exerted a synergistic and prolonged inhibition of HBsAg ([~]4log10 IU/mL, vs PBS) and induced a higher incidence of HBsAg seroconversion ([~]30%), comparing with either monotreatment. Mechanistically, combined therapy improved the functionality of global T and B cells, triggered increased anti-HBs producing B cells, and enhanced IFN{gamma}-producing T cells. scRNA-seq analysis revealed that the combined therapy reduced inhibitory B cell-B cell interaction, enhanced MHC-I signaling mediated T cell-T cell communication, and improved T cell-B cell crosstalk, thus improving the functionality of T and B cells. Enhanced MHC-II signaling networks across B cells and hepatocytes/Cd8+ T cells further promoted HBsAg seroconversion in the combined treatment groups. These results together provided scientific rationale and lessons for the combination of the two towards better therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/626539v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@84785aorg.highwire.dtl.DTLVardef@f984fborg.highwire.dtl.DTLVardef@189a966org.highwire.dtl.DTLVardef@d081f9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIShutting down HBsAg through RNA interference augmented the antiviral immune effects of PEGIFN in chronic HBV-carrier IFNAR-hEC mice. C_LIO_LICombined RNAi plus PEGIFN augmented the functionality of T cells, promoted B cell activation and class switch, but also exerted some suppressive effects on B cells. C_LIO_LIReduced inhibitory B cell-B cell interaction, enhanced MHC-I signaling between T cells and T cells, and improved T cell-B cell crosstalk, improved the functionality of T cells and B cells. C_LIO_LIEnhanced MHC-II signaling networks across B cells and hepatocytes/ Cd8+ T cells further promoted HBsAg seroconversion in RNAi plus PEGIFN combined treatment groups. C_LI

microbiology↗