Biology subjects

Lu, R.

Publications and source records attributed to Lu, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Redox-dependent extracellular interaction networks of Cysteine-Rich Receptor-Like Kinases

Reactive oxygen species (ROS) regulate plant development and immunity, but how extracellular ROS signals are decoded and whether the Cysteine-rich Receptor-like Kinases (CRKs) truly serve as the long-suspected ROS sensors remains uncertain. Here, we combine high-throughput interactomics, redox proteomics, structural modelling, and genetics to map a ROS-dependent CRK interaction landscape in Arabidopsis thaliana. Using a redox-dependent interactome assay (RIACRK) on 40 CRK extracellular domains (ECDs), we identified ROS-modulated dimerisation networks with enhanced inter-community connectivity and hub redistribution in the presence of ROS. Integrating this with developmental and flg22-induced expression profiles reveals spatiotemporally limited subnetworks that likely function during activated immunity and leaf senescence, both of which are associated with extensive ROS production. Differential cysteine alkylation coupled with mass spectrometry shows that cysteines in a subset of CRK ectodomains undergo ROS-dependent oxidation. Notably, solvent-exposed, vicinal cysteines C228/C229 in CRK28 emerge as prime redox-sensitive candidates. CRK28 homodimerises and heterodimerises with CRK17 in vivo, and mutation of C228/C229 retains plasma membrane localisation but abolishes CRK28 homodimerisation, indicating a redox-controlled dimerisation switch. Loss of CRK28 delays senescence, while CRK28 overaccumulation from its native promoter causes dwarfism, premature senescence, autoimmune-like phenotypes, extensive phosphoproteome rewiring, and associations with Pathogenesis Related (PR) proteins, ROS-detoxifying enzymes, receptor(-like) kinases, and vesicle trafficking components. These results indicate that CRK28 is a potential ROS-regulated hub connecting extracellular redox signals to CRK network organisation, immune response, and age-related senescence.

plant biology↗

bioRxiv2026

Neural Arming Niche in Tumor-Draining Lymph Nodes Programs CD8⁺T Cell Cytotoxicity via GZMB Norepinephrinylation

Lymph nodes are densely innervated neuro-immune hubs where naive T cells are primed and "armed" with cytotoxic machinery, yet how local neural cues in tumor-draining lymph nodes (tdLNs) set CD8 T cell effector reserves remains unclear. Here we show that tdLN-local sympathetic norepinephrine (NE) instructs CD8 T cell cytotoxic capacity by stabilizing the granzyme B (GZMB) pool through covalent norepinephrinylation. Exercise, used as a physiological perturbation, increases GZMB selectively in tdLN and tumor CD8 T cells without altering T cell abundance or cytokine output; tdLN sympathectomy abolishes these effects and reduces tdLN NE enrichment. Mechanistically, NE enters CD8 T cells and norepinephrinylates GZMB at Gln43, limiting UHRF1-mediated ubiquitination and proteasomal degradation. Genetic attenuation of norepinephrinylation destabilizes GZMB, impairs cytotoxicity, and accelerates tumor growth. Brief ex vivo NE conditioning increases GZMB reserves and improves adoptive tumor control.

cancer biology↗

bioRxiv2026

QuantCell: machine learning based cell annotation from qualitative and quantitative imaging profiles

Recent advances in spatial omics enable high-resolution, multiplexed imaging of RNA and protein expression, but cell annotation remains challenging, particularly in complex tissues with numerous markers or rare cell types. Here, we present QuantCell, a machine learning framework that leverages quantitative imaging data to improve annotation derived from qualitative profiles. QuantCell evaluates multiple models and applies a user-defined false discovery rate to ensure high-confidence annotation. Using PhenoCycler imaging of mouse bone marrow, QuantCell increased annotated cells from 33.1% to 90.2% at 5% FDR, achieving 96.5% accuracy. QuantCell supports diverse imaging platforms and robustly detects rare cell populations.

bioinformatics↗

bioRxiv2026

Tracking Human HSCs and MPPs in Mice Reveals Distinct Clonal Dynamics and Responses to Pre-transplant Conditioning

Blood and immune cell regeneration is sustained by hematopoietic stem and progenitor cells (HSPCs), which form the therapeutic basis of bone marrow transplantation. While the functional hierarchy of mouse HSPC subsets is well characterized, the distinct roles of human HSPC populations remain less well defined, particularly at clonal resolution and in the context of transplantation conditioning. While clonal tracking in humans and non-human primates has significantly advanced our understanding of hematopoietic dynamics, prior studies predominantly focused on CD34+ cells, a heterogeneous population of HSPCs. Moreover, secondary transplantation is considered the gold standard for distinguishing hematopoietic stem cells (HSCs) from multipotent progenitors (MPPs) in mice, but it has not been effectively utilized to study human HSPC populations. To address this knowledge gap, we performed quantitative clonal tracking of purified human HSCs (hHSCs) and human MPPs (hMPPs) in NSGW41 mice across primary and secondary transplantation under no conditioning, busulfan, and irradiation. Consistent with prior studies, both hHSCs and hMPPs sustained long-term multilineage reconstitution and differed in engraftment rates. Our quantitative clonal analysis further revealed that hHSC clones generated more blood cells, initiated lymphoid production earlier, and exhibited more robust multilineage differentiation than hMPP clones. hHSC clones were also less sensitive to conditioning, maintaining stable lineage biases. Notably, busulfan and irradiation differentially affected the magnitude, lineage bias, and timing of hematopoietic reconstitution without altering engraftment. During secondary transplantation, hHSCs and hMPPs contributed comparably to hematopoietic reconstitution, but their overall output, particularly monocytes and T cells, was substantially reduced. In contrast to primary recipients, human chimerism of secondary recipients in the peripheral blood was diminished relative to the bone marrow and spleen, and more hHSPC clones contributed to hematopoiesis. Extramedullary hematopoiesis was observed in all secondary recipients, with comparable contributions from hHSC and hMPP clones. Overall, this study provides insights into the distinct functions of hHSCs and hMPPs, the influence of conditioning, and the inefficiency of human hematopoiesis through serial transplantation. These findings advance our understanding of human hematopoiesis and provide a framework for utilizing and optimizing experimental models, improving transplantation conditioning strategies, and informing the preclinical evaluation of HSC-based cell and gene therapies.

cell biology↗

bioRxiv2026

Telomere length of both parents contribute to heritable POT1 cancer-predisposition syndrome

Germline mutations in POT1 are linked to familial cancer predisposition, and somatic POT1 mutations occur recurrently in tumors. These mutations promote oncogenesis by enabling aberrant telomere elongation. For inherited POT1 mutations, a critical question is the extent to which elongated telomeres are transmitted to the next generation from the POT1 carrier parent and whether the inherited excessively long telomeres elevate cancer risk. Using a nanopore sequencing approach that provides haplotype-specific telomere length measurements, we examined telomere inheritance in families harboring POT1 mutations. We found that individuals preferentially inherit their longest telomeres from the carrier parent, consistent with extensive telomere elongation in the carrier germline, whereas comparatively short telomeres are predominantly inherited from the non-carrier parent. Analysis of carrier and non-carrier siblings further showed that telomeres inherited from both parents are elongated in POT1 carriers, with the shortest telomeres undergoing preferential elongation. These findings support a potential mechanism of genetic anticipation in which POT1 mutations progressively reduce the likelihood that short telomeres capable of enforcing telomere-based tumor suppression are inherited from the carrier parent. Together, our results demonstrate that telomeres inherited from both parents jointly shape telomere-based tumor suppressive barriers. Summary sentenceAllele specific nanopore sequencing reveals that POT1 mutations reshape germline and somatic telomere dynamics, uncovering a novel mechanism of generational anticipation driven by preferential elongation of short inherited telomeres.

genetics↗

bioRxiv2026

GP130 Y814 SIGNALING IS REQUIRED FOR THE DYNAMIN-MEDIATED ENDOCYTOSIS, MAPK/P38 ACTIVATION AND PERSISTENCE OF CHRONIC SYSTEMIC INFLAMMATION INDUCED BY HIGH FAT DIET

In chronic inflammatory diseases such as obesity, tissue and cellular homeostasis are disrupted by persistent low-grade inflammation, where one of the most prominent pro-inflammatory cytokines that correlates with age and body mass index (BMI) is IL-6. All members of the IL-6 family of cytokines signal through their obligate co-receptor gp130, in whichsignaling tyrosines on the intracellular portion of gp130 activate multiple downstream pathways such as the canonical STAT and MAPK pathways. However, non-canonical gp130 pathways such as SRC family of kinases (SFKs) signaling have emerged as drivers of cellular stress response. Our recently published mutant mouse model carrying a constitutive inactivation of gp130-Y814 (F814 mice), which impairs SFK activation, exhibited an enhanced resolution of inflammatory responses and improved regenerative outcomes in both acute skin wound healing and post-traumatic osteoarthritis models. The current study was designed to explore whether the gp130-Y814 mutation reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model and to interrogate the possible downstream cellular mechanisms that are affected. In response to HFD, F814 mice showed significantly reduced systemic and tissue-specific inflammatory responses and protection from obesity-induced bone loss and osteoarthritis compared to wild type (WT) mice. After extensive characterization, the role of gp130-Y814 in monocytes/macrophages appeared to be dispensable, but we discovered that the F814 mutation blunts gp130 receptor internalization, p38/MAPK activation and the release of matrix metalloproteinases (MMPs) in chondrocytes. Finally, we showed that F814 chondrocytes had markedly reduced activation of the SFK-dependent GTPase dynamin 2 (Dyn2) in response to catabolic IL-6 cytokines. Introduction of a dominant-negative Dyn2 mutant into WT chondrocytes phenocopied the effects of the F814 mutation, resulting in attenuated p38/MAPK signaling and reduced MMP activation following stimulation with IL-6 cytokines. This study demonstrates, for the first time, that the Y814 residue is directly implicated in Dyn2-mediated internalization of the gp130 receptor, thereby modulating downstream signaling and contributing to pathological outcomes in chronic inflammatory and degenerative diseases in a cell type-specific manner.

cell biology↗

bioRxiv2025

A programmable mRNA platform for miRNA detection via miRNA-mRNA2 triplex-mediated ribosomal frameshifting

Programmed -1 ribosomal frameshifting (-1 PRF) is a recoding mechanism utilized by viruses to expand their coding capacity and modulate the stoichiometric ratio of -1 frame and 0 frame translation products. The stability of mRNA secondary structure at the ribosomal entry site within the frameshifting stimulating elements (FSEs) determines the frameshifting efficiency. Here, we report the development of a programmable mRNA-based platform that detects specific mature microRNA (miRNA or miR) by converting their presence into a quantifiable protein output through miRNA-triggered -1 PRF. We designed a triplex-forming mRNA (TF-mRNA) platform to selectively trap target miRNAs through the formation of major-groove mRNA-miRNA-mRNA (miR-mRNA2) triplexes. Bio-layer interferometry and fluorescence binding studies confirmed that TF-mRNA forms stable complexes with cognate miRNAs with low nanomolar affinity and prolonged dissociation rate. Critically, the formation of miR-mRNA2 triplex robustly stimulated ribosomal frameshifting in a cell-free dual-luciferase translation system, acting as a miRNA-dependent molecular switch. The generality of this TF-mRNA platform has been verified for several disease-associated purine-rich miRNAs, and it is suitable for targeting a wide range of other purine-enriched miRNAs. This programmable TF-mRNA platform establishes a foundation for developing novel diagnostic tools and synthetic biology circuits that convert the presence of miRNA into a quantifiable protein output. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/690164v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d6d9f7org.highwire.dtl.DTLVardef@8008e6org.highwire.dtl.DTLVardef@1c8344borg.highwire.dtl.DTLVardef@e153ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Neural dynamics in ventrolateral prefrontal cortex underlie learning from feedback

Learning often depends on feedback, yet how positive and negative outcomes reorganize target representations to support later memory retrieval remains poorly understood. Accumulating evidence suggests that the ventrolateral prefrontal cortex (vlPFC) acts as a central hub linking learning and retrieval, raising the possibility that it plays a critical role in this process. Here we analysed spiking activity and local field potentials (LFPs) recorded from vlPFC while monkeys performed a multi-cycle object-learning task. During the initial learning cycle, correct and incorrect feedback elicited distinct vlPFC neural responses in both spiking and LFPs. In particular, positive feedback produced elevated theta power and enhanced phase-amplitude coupling (PAC) between theta phase and high-frequency amplitude, associated with sustained suppression of neural spiking. Incorrect feedback induced stronger beta power. Despite comparable levels of object information under both feedback conditions, decoders trained and tested within the same feedback state outperformed those tested across states, revealing feedback-dependent coding formats. State-space and cross-period generalisation analyses further showed that object representations following positive feedback were geometrically closer to and shared a common coding format with those reinstated during later retrieval, indicating that feedback reshapes neural geometry toward retrieval-compatible states. Moreover, these geometric and generalisation effects were selectively expressed on electrodes showing stronger PAC or beta power, suggesting that oscillatory coordination may regulate how feedback signals are transformed into stable target codes. Together, our results reveal how vlPFC serves as a critical bridge between learning and memory retrieval, with feedback-driven dynamics reorganizing population geometry through rhythmic coordination and bringing successful outcome states closer to future retrieval representations.

neuroscience↗

bioRxiv2025

Retinal ganglion cell input to superior colliculus encodes salient information

The superior colliculus (SC) is critical in detecting and responding to salient stimuli. However, it is unknown whether salience signals are inherited from external sources such as retina and cortex, or arise from the intrinsic circuitry within SC. We used in vivo 2-photon calcium imaging with adaptive optics to examine the activity of retinal ganglion cell (RGC) axon terminals in superficial SC (sSC). We found that RGC boutons exhibit similar saliency-related activity as sSC neurons. A majority of boutons respond more strongly to salient discontinuities of visual features, and change their orientation preference depending on stimulus properties such as the orientations of edges of drifting gratings. We furthermore demonstrate that these effects arise from the center-surround receptive field structure of RGCs. Our results show that the orientation tuning of RGCs is highly flexible, and that saliency encoding originates in retina rather than sSC.

neuroscience↗

bioRxiv2025

Engineering a Biomimetic Multiphasic Suture Anchor System for Enhanced Rotator Cuff Enthesis Regeneration

Conventional suture anchor methods in rotator cuff repair often fail to replicate the native tendon-to-bone interface, leading to re-tears due to stress concentrations and poor biological integration at anchor sites. To address these challenges, we engineered a biomimetic multiphasic scaffold system (BMS) that integrates with standard suture anchors and deliver spatially organized structural and biological cues to enhance enthesis regeneration. The BMS comprises three distinct phases: aligned nanofibrous decellularized bovine Achilles tendon extracellular matrix (dECM) with stiff methacrylated hyaluronic acid (MeHA) for tendon regeneration; nonaligned nanofibrous dECM with soft MeHA for fibrocartilage formation; and a porous, citrate-based composite scaffold with bioactive glass for bone integration. In vitro, the BMS facilitated zone-specific tenogenic, fibrochondrogenic, and chondrogenic differentiation. Further, in vivo, it promoted successful integrative healing, forming distinct tendon, fibrocartilage, and bone regions at the repair site. This advanced multiphasic scaffold replicates native tissue properties, offering a promising strategy to improve rotator cuff repair. Its integration with conventional suture anchors provides an innovative design that enhances mechanical fixation and guides enthesis healing to reduce re-tear rates. Broadly, this platform offers a versatile solution for biointegrative repair strategies across complex soft-to-hard tissue interfaces.

bioengineering↗

bioRxiv2025

Discovery of Novel Probiotic Species to Improve Infant Health

Selecting an appropriate infant formula can be challenging for parents given the wide range of available options, which may lead to suboptimal choices or frequent switching. Our recent studies in mice demonstrated that the maternal microbiota--particularly Limosilactobacillus reuteri present in breast milk--plays a critical role in shaping neonatal immunity by promoting the production of immunoglobulin A (IgA), a key antibody for defense against enteric pathogens. To extend these findings to humans, we analyzed fecal samples from healthy breastfed infants using shotgun metagenomic sequencing combined with whole-bacterial-cell ELISA to assess associations between specific taxa and pathogen-reactive IgA. We found that members of the Oscillibacter species (including Oscillibacter sp. KLE1728, KLE1745, PC13, PEA192, and O. valericigenes) and Morganella (e.g., M. morganii, Morganella sp. EGD-HP17, and Morganella sp. HMSC11D09) were strongly correlated with IgA responses against Salmonella and Shigella. In contrast, IgA reactive to enterohemorrhagic Escherichia coli (EHEC) was associated with other genera, including Staphylococcus. Notably, Lactobacillus species (L. bombi, L. kefiri, L. equi, and L. rhamnosus) were consistently linked to elevated IgA responses against Salmonella, Shigella, and EHEC. Moreover, levels of anti-Salmonella and anti-Shigella IgA were positively correlated with the use of infant formula containing prebiotic fibers or human milk oligosaccharides (5-HMO). Collectively, these findings highlight novel commensal taxa with potential as probiotic candidates to guide the development of next-generation infant formulas aimed at enhancing mucosal immunity and supporting infant health. ImportanceEarly-life nutrition shapes immune maturation, yet the microbial factors driving mucosal antibody responses remain poorly defined. Through integrated metagenomic and IgA profiling, this study identifies commensal taxa, particularly Lactobacillus, Oscillibacter, and Morganella, associated with pathogen-specific IgA in infants. These taxa may enhance mucosal defense against enteric pathogens, revealing key microbial contributors to early immune development. The findings provide a foundation for designing next-generation infant formulas that leverage targeted probiotics or prebiotics to promote protective IgA responses and gut health.

microbiology↗

bioRxiv2025

Characterizing the role of RSD-6 in the biogenesis of virus-derived small interfering RNAs and the modulation of viral pathogenesis

Small interfering RNAs (siRNAs) produced through the processing of viral double-stranded RNAs mediate potent antiviral RNA interference (RNAi) in eukaryotes. In Caenorhabditis elegans, such an antiviral defense is further amplified through the production of secondary siRNAs, yet the mechanisms by which secondary virus-derived siRNAs (vsiRNAs) confer protection remain poorly understood. Here, we characterize the role of rsd-6, which encodes a Tudor domain protein and plays important role in antiviral RNAi, in vsiRNA biogenesis and modulation of viral pathogenesis. Using CRISPR Cas9-generated rsd-6 null mutants, we show that both primary and secondary vsiRNAs accumulate normally in the absence of RSD-6, indicating that it functions downstream of secondary vsiRNA biogenesis. We further showed that secondary vsiRNAs generated in rrf-1-independent manner remained detected in the absence of RSD-6 and viral replication is further enhanced in rrf-1;rsd-6 double mutants compared to rrf-1 single mutants, suggesting a role of rsd-6 in mediating antiviral guided by all secondary vsiRNAs. Consistently, rsd-6 mutants exhibited more severe pathogenesis upon Orsay virus infection compared to rrf-1 mutants, underscoring its role as a major determinant of viral disease outcome. Domain characterization established that the N-terminal tandem domains of RSD-6 are required for antiviral activity, while the C-terminal Tudor domains are dispensable. Functional conservation was confirmed in C. briggsae, where silencing of the rsd-6 homolog enhanced viral replication. Together, our findings identify RSD-6 as a key effector acting downstream of secondary vsiRNA production and highlight its conserved role in modulating viral replication and pathogenesis across Caenorhabditis species. ImportanceIn C. elegans, the RNAi-mediated antiviral defense relies on the production of secondary virus-derived siRNAs (vsiRNAs) to achieve an amplified antiviral effect. However, the mechanism by which these secondary vsiRNAs confer protection remains poorly understood. This is primarily due to the limited number of identified key effector genes. To address this knowledge gap, we profiled vsiRNA biogenesis in loss-of-function mutants and discovered that rsd-6 is dispensable for the production of secondary vsiRNAs, suggesting a role of rsd-6 in mediating antiviral defense downstream of secondary vsiRNA biogenesis. Worm survival assay further confirmed that rsd-6 is a critical modulator of viral pathogenesis and its antiviral function is conserved across Caenorhabditis species. The RSD-6 protein features three N-terminal tandem domains of unknow function and two tandem Tudor domains at its C-terminus. Our domain analyses demonstrated that the N-terminal tandem domains, but not the C-terminal Tudor domains, are essential for antiviral function. The identification of rsd-6 as a key effector gene acting downstream of vsiRNA biogenesis provides a solid foundation for elucidating the mechanism of antiviral RNAi amplification.

immunology↗

bioRxiv2025

MDS-associated SF3B1 mutations promote aberrant fate choice of hematopoietic stem cell via mis-splicing of mediator kinase module component CDK8

Mutations in RNA splicing factor SF3B1 are among the most common in MDS and are strongly associated with MDS with ring sideroblasts (MDS-RS). While aberrant splicing of terminal erythroid regulators has been implicated in MDS pathogenesis, the impact of SF3B1 mutations on early hematopoietic progenitor function remains unclear. Here, we identify CDK8, a key kinase of the mediator complex involved in transcriptional regulation, as a recurrent mis-spliced target in SF3B1-mutant MDS. Mutant SF3B1 induces cryptic 3' splice site selection in CDK8, leading to loss of CDK8 mRNA and protein. Using primary human HSPCs, our study identifies CDK8 as an important regulator of HSPC homeostasis and cell fate determination. CDK8 depletion results in expansion of HSPCs and shifts differentiation toward the erythroid and myeloid lineages, mirroring phenotypes observed in SF3B1-mutant MDS. Lastly, functional restoration of CDK8 rescues early erythroid phenotypes in SF3B1-mutant cells. These findings implicate CDK8 mis-splicing as a mechanistic driver of altered progenitor fate and dysplasia in SF3B1-mutant MDS, linking aberrant splicing to transcriptional dysregulation and hematopoietic lineage commitment.

cancer biology↗

bioRxiv2025

A new dual-affinity peptide nucleic acid for targeting miRNA-21 precursor rescues tumor repressor PTEN expression

MicroRNAs (miRNAs) have multiple functions in cells and are related to many diseases including cancer by regulating posttranscriptional gene expression. The microRNA precursors (pre-miRs), which can be cleaved by Dicer endonuclease to produce mature miRNAs, often have a cleavage site consisting of both double-stranded (ds) and single-stranded (ss) RNA structures. Peptide nucleic acid (PNA) is a kind of analogue of DNA which can hybridize to DNA or RNA through Watson-Crick and Hoogsteen pairing. We previously reported a novel dual-affinity PNA (daPNA) platform that can simultaneously form a duplex and a triplex with a target RNAs ssRNA-dsRNA junction region. The PNA-RNA complex structure is stabilized by forming antisense PNA (asPNA)-ssRNA duplex immediately adjacent to a chemically modified dsRNA-binding PNA (dbPNA)-dsRNA triplex. In this study, we further explored the application of the daPNA platform to target the precursor of miR-21, which is considered an oncogene. We have designed a set of PNAs including asPNAs, dbPNAs, and daPNAs. The nondenaturing polyacrylamide gel electrophoresis (PAGE) and biolayer interferometry (BLI) data reveal that daPNA-21-10 can strongly bind to pre-miR-21 with high specificity. The data show that pre-miR-21 is easily targeted by traditional antimir, asPNA or dbPNA and optimization of the dsRNA-ssRNA junction position is needed for identifying a tightly binding daPNA. Furthermore, daPNA-21-10 not only inhibits the Dicer activity on pre-miR-21 in cell-free assays, but also down-regulates the expression of miR-21, rescuing PTEN protein expression in cells. Taken together, the biofunction and programmability of daPNA make it a promising platform for probing and regulating miRNA biogenesis and many other RNA-involved biological processes. HighlightsO_LISimultaneous recognition of dsRNA and ssRNA regions C_LIO_LIRNA targeting by dividing and conquering through a dsRNA-ssRNA junction construction C_LIO_LISubstrate-specific inhibition of Dicer acting on pre-miR-21 C_LIO_LIDerepressing PTEN expression by inhibiting miR-21 maturation C_LI O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/668835v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@c8af64org.highwire.dtl.DTLVardef@172d2bdorg.highwire.dtl.DTLVardef@1698ea1org.highwire.dtl.DTLVardef@112fa16_HPS_FORMAT_FIGEXP M_FIG C_FIG eToc BlurbLian et al. show that the miR-21 precursor structure containing the Dicer cleavage site can be targeted by a new type of dual-affinity peptide nucleic acids (daPNAs) through the creation of a dsRNA-ssRNA junction, energetically optimized for the simultaneous duplex and triplex formation. Pre-miR-21 structure, which is hardly accessible to traditional antisense strategies and triplex formation alone, can be targeted by a daPNA with high sequence/structure specificity and strong binding affinity. daPNA platform has a great potential in probing many other RNA structures and broad therapeutic applications.

molecular biology↗

bioRxiv2025

Connectomic reconstruction from hippocampal CA3 reveals spatially graded mossy fiber inputs and selective feedforward inhibition to pyramidal cells

The mossy fiber (MF) connections to pyramidal cells in hippocampal CA3 are hypothesized to participate in pattern separation and memory encoding, yet no large-scale neuronal wiring diagram exists for these connections. We assembled a 3D electron microscopy volume ([~]1x1x0.1mm3) from mouse hippocampal CA3. By proofreading and automated segmentation, we reconstructed and classified all soma-containing neurons--including 1,815 pyramidal cells and 229 inhibitory cells--and over 55,000 MFs. Pyramidal cells receive more numerous MF inputs along a proximodistal gradient. Some distal cells show surprisingly high convergence via relatively small terminals with fewer vesicles. Pyramidal cells share significantly more MF inputs than networks randomized by degree-preserving swap, and are better approximated by networks randomized by proximity-preserving swap. We identify a feedforward inhibitory circuit from MFs via perisomatic interneurons that selectively target a pyramidal subtype. We demonstrated large-scale mapping across levels in the hippocampus--from circuits to cell types to vesicles. The dataset is shared through Pyr, an online platform for hippocampal connectomics.

neuroscience↗

bioRxiv2025

Towards the Development of Isoenergetic Peptide Nucleic Acid Based Probes Targeting Double-Stranded RNAs Through Enhancing Sequence-Specific Stacking Interactions

Peptide nucleic acid (PNA), a synthetic nucleic acid analog, exhibits substantial potential in biotechnology and therapeutic applications due to its high binding affinity and nuclease/protease resistance. Chemically modified PNAs capable of forming stable triplex structures with double-stranded RNAs (dsRNAs) under near-physiological conditions further expand their utility by enabling sequence-specific precise targeting and probing of functional RNA structural motifs. However, the presence of inverted Watson-Crick pairs (C-G and U-A) may significantly weaken the triplex formation of the dsRNA-binding PNAs (dbPNAs). Our previous work demonstrated that dbPNA P3 (composed of L, T, and Q monomers for the recognition of G-C, A-U, and C-G base pairs, respectively) can stimulate ribosomal frameshifting by binding to rHP2, a model RNA hairpin structure in an mRNA, albeit with suboptimal efficiency, due to its significantly weakened Q*C-G triple formation. We hypothesize that incorporating s2U adjacent to Q may offer unique stacking and hydrogen bonding interactions facilitating the development of isoenergetic dbPNA-based probes binding toward dsRNAs with varied sequences. In this study, we investigate how incorporating s2U adjacent to Q residues in P3 influences its binding to rHP2. Bio-layer interferometry (BLI) and non-denaturing polyacrylamide gel electrophoresis (PAGE) analyses demonstrate that substituting T with s2U at the N-terminal position adjacent to Q (P3-2QT) enhances binding affinity by [~]10-fold compared to unmodified P3, whereas C-terminal substitution (P3-TQ2) yields only a 2-fold improvement. Consistent with these findings, a cell-free dual-luciferase reporter assay reveals that P3-2QT significantly increases ribosomal frameshifting efficiency compared to P3 and P3-TQ2. Molecular dynamics simulations further indicate that P3-2QT maintains enhanced PNA-PNA stacking stability, particularly between s2U3 and Q4, suggesting a structural basis for its superior activity. Intriguingly, analogous s2U substitution in the P5 oligomer with the Q replaced by L does not confer a comparable enhancement in binding to the target RNA (rHP1) and frameshifting stimulation, highlighting the context-dependent nature of this modification. To assess the broader applicability of the s2U-Q motif, we examined its effect in dbPNAs targeting the RNA panhandle structure of influenza virus A and precursor microRNA-21, respectively. Both PAGE and BLI data confirm that s2U incorporation improves binding affinity, reinforcing the generality of this strategy. These findings underscore the potential of sequence-dependent uracil thiolation in optimizing triplex-forming dsRNA-binding PNAs, warranting further exploration of modified nucleobase designs to enhance their binding and functional properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/662161v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15d86aborg.highwire.dtl.DTLVardef@1347369org.highwire.dtl.DTLVardef@852040org.highwire.dtl.DTLVardef@84b0a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThiolation of uracil upstream of Q base in PNA markedly enhances triplex binding affinity and enables isoenergetic and orthogonal targeting/activation. C_LIO_LIN-terminal s2U modification adjacent to Q residues increases PNA-dsRNA binding by [~]10-fold and significantly boosts ribosomal frameshifting efficiency. C_LIO_LIBiophysical assays and molecular dynamics simulations reveal that thiolation improves PNA stacking stability and slows dissociation, thereby enhancing functional activity toward structured RNA targets. C_LIO_LIThe general applicability of s2U modification is demonstrated with influenza A virus RNA panhandle structure and precursor micoRNA-21 targets, underscoring its broad potential for optimizing PNA-based therapeutics and biotechnological tools. C_LI

biochemistry↗

bioRxiv2025

CaV1.2-dependent excitation-transcription coupling modulates nociception

Peripheral nociceptive sensory neurons integrate various noxious inputs, resulting in local depolarization that triggers the firing of action potentials and thus the sensation of pain. We recently reported that nociceptor depolarization itself initiates signaling by the calcium channel Cav1.2 causing acute hyperalgesia in vivo. However, whether this mechanism initiates excitation-transcription (E-T) coupling and thereby leads to long-lasting modulation of nociceptor activity remains poorly understood. Using high content imaging of dorsal root ganglion (DRG) neurons, we here found that depolarization of nociceptors induces phosphorylation of the transcription factor (TF) cAMP-response element binding protein (CREB), which was affected by inhibition of protein kinase A (PKA) and calcineurin, but not Ca2+/calmodulin-dependent protein kinases. Genetic deletion or pharmacological inhibition of Cav1.2 confirmed its role in calcium-dependent kinase signaling and CREB phosphorylation after depolarization. In line with this, pharmacological modulation of Cav1 channels affected the expression of a subset of depolarization-regulated immediate early genes known to orchestrate a broader transcriptional response. Indeed, RNA-Seq analysis of DRG neurons from mice with a tissue-specific deletion of Cav1.2 in nociceptive sensory neurons (SNS-Cacna1c-/- mice) revealed downregulation of multiple calcium and potassium channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion. Furthermore, repetitive firing of action potentials and release of the neuropeptide CGRP was impaired in Cav1.2-deficient sensory neurons. SNS-Cacna1c-/- mice showed increased sensitivity to noxious heat and exacerbated inflammatory but not neuropathic pain. In conclusion, our data suggest a Cav1.2-dependent E-T coupling mechanism in nociceptors that counteracts nociception in vivo. HighlightsO_LIDepolarization induces Cav1.2- and PKA-dependent phosphorylation of the transcriptional regulator CREB in nociceptors C_LIO_LICav1.2 regulates the expression of depolarization-induced immediate early genes and induces reorganization of the signaling network in nociceptors C_LIO_LICav1.2-deficient nociceptors downregulate multiple ion channel subunits as well as proteins involved in synaptic vesicle release and cell adhesion C_LIO_LICav1.2-deficient nociceptors show impaired depolarization-induced PKA-II signaling, CREB phosphorylation, repetitive firing of action potentials, and release of the neuropeptide CGRP C_LIO_LINociceptor-specific Cav1.2-deficiency in mice increases the sensitivity to noxious heat and exacerbates inflammatory pain C_LI In briefIsensee et al. report that depolarization of nociceptors induces calcium influx through Cav1.2 channels, thereby initiating a signaling cascade that links neuronal excitation to transcriptional regulation of immediate early genes (E-T coupling). Nociceptor-specific knockout of Cav1.2 led to transcriptional changes in nociceptor-specific genes and was associated with impaired depolarization-induced signaling, action potential firing, and neuropeptide release. As the Cav1.2-deficient mice show increased noxious heat sensitivity and exacerbated inflammatory pain, these findings suggest that Cav1.2-dependent E-T coupling mechanism counteracts nociception in vivo.

neuroscience↗

bioRxiv2025

Isoform-Specific Roles of NTRK2 in Pulmonary Vascular Regeneration

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants with no curative therapy, characterized by impaired alveologenesis and capillary formation. However, the molecular mechanisms underlying endothelial dysfunction, a key driver of BPD pathogenesis in human, remain poorly understood. Here, through multiomic profiling of vascular endothelial cells isolated from control and BPD patient lungs, we uncovered an expansion of general capillary endothelial cells (gCap) with aberrant expression of the neurotrophic receptor tyrosine kinase 2 (NTRK2) in BPD. Importantly, we identified a pathological NTRK2 isoform switch that dictates the regenerative capacity of gCap cells. Full-length NTRK2 (NTRK2-FL) promoted gCap regeneration in response to hyperoxic injury, whereas RBFOX2-mediated splicing of NTRK2-FL into a truncated isoform (NTRK2-T1) contributed to maladaptive responses and irreversible alveolar simplification in severe BPD cases. Restoring NTRK2-FL using lipid nanoparticle-delivered mRNA promoted angiogenesis and reversed alveolar simplification in vessel organoids and BPD-like mice. These findings identified NTRK2 isoform imbalance as a key driver of endothelial dysfunction and support isoform-specific RNA therapy as a promising strategy for vascular regeneration and repair. HIGHLIGHTS* Multiomic and spatial profiling reveal abnormal gCap subtype in human BPD lungs * NTRK2 isoform switch dictates endothelial regeneration in response to hyperoxic injury * RBFOX2 mediates splicing from NTRK2-FL to NTRK2-T1, driving irreversible alveolar damage * NTRK2-FL mRNA therapy restores vascular regeneration after injury

developmental biology↗