bioRxiv : the preprint server for biology最新文献

Single-molecule microscopy has been widely used to study the structure and dynamics of RNA, but extension to larger systems such as long non-coding RNA (lncRNA) has proven challenging. The length and complex nature of lncRNA limit the techniques that can be readily used to study these aspects, but methods such as single-molecule kinetic analysis of RNA transient structure (SiM-KARTS), where the binding of a short, complementary oligonucleotide probe is used to determine accessibility of a specific region of the RNA, are promising. However, adapting SiM-KARTS to systems as complex as lncRNA requires careful optimization of experimental variables that have not been thoroughly explored. In this work, SiM-KARTS, thermal denaturation experiments, and circular dichroism spectroscopy were used to analyze the binding behaviors of probes with alternative backbone chemistries, specifically DNA with locked nucleic acid residues incorporated and morpholinos. A segment of lncRNA that enabled control over the accessibility of the target sequence was used as a model. We show that optimizing probe backbone chemistry can allow for a more precise distinction between different structures of the target RNA, and for fine-tuning of probe binding stability without the structural impacts that other variables such as ionic concentration may have. These results provide design principles for the application of SiM-KARTS to target RNAs of increased complexity such as lncRNA.

Therapeutic peptides occupy a unique middle ground in drug discovery, offering the high specificity of protein interactions with the chemical diversity of small molecules, yet they currently fall in a computational blind spot. Existing AI tools cannot handle them effectively: protein models are restricted to natural amino acids, while chemical models struggle to process large, polymer-like sequences. This disconnect has forced the field to rely on static chemical descriptors that fail to capture subtle chemical details. To bridge this gap, we present PeptideCLM-2, a chemical language model trained on over 100 million molecules to natively represent complex peptide chemistry. PeptideCLM-2 consistently outperforms both chemical descriptors and specialized AI models on critical drug development tasks, including aggregation, membrane diffusion, and cell targeting. Notably, we find that when model parameters reach the 100 million scale, the transformer architecture is able to learn chemical properties from molecular syntax alone.

Large-conductance Ca ²⁺ - and voltage-activated K ⁺ (BK) channels are widely expressed, including in the brain where they shape neuronal excitability. Their physiological functions are strongly influenced by cell-type-specific auxiliary subunits. The auxiliary γ3 subunit (LRRC55) enhances BK-channel activation by shifting voltage-dependent gating toward more negative potentials; however, its protein distribution and in vivo function remain unclear. Here, we generated knock-in mice carrying a C-terminal epitope tag on endogenous LRRC55 to map its expression, and Lrrc55 knockout mice to test its function. LRRC55 protein was selectively enriched in cerebellar Purkinje cells. Lrrc55 deletion produced ataxia-like impairments in gait, balance, and coordination. In acute slices, pharmacological BK-channel block with paxilline altered Purkinje cell simple- and complex-spike firing in wild-type mice, whereas these BK-dependent effects were largely absent in Lrrc55 knockouts, indicating that LRRC55 is required for BK channels to shape Purkinje cell firing under these conditions. Moreover, LRRC55 loss disrupted cerebellar synaptic plasticity, abolishing parallel fiber-Purkinje cell long-term potentiation and eliminating climbing fiber-Purkinje cell long-term depression, phenocopying paxilline in wild-type cells. Together, these results identify LRRC55 as a Purkinje-cell-enriched auxiliary subunit that is essential for BK-dependent excitability and plasticity and that supports normal cerebellar motor function.

Significance statement: BK channels are important regulators of neuronal firing, but how they are modulated in specific brain regions is poorly understood. We show that the BK γ3 subunit LRRC55 is selectively enriched in cerebellar Purkinje cells and is required for normal motor coordination. Loss of LRRC55 removes BK-dependent modulation of Purkinje cell firing and abolishes two major forms of cerebellar synaptic plasticity, parallel fiber long-term potentiation and climbing fiber long-term depression, while phenocopying pharmacological BK-channel inhibition. These findings reveal an in vivo, cell-type-specific mechanism by which an auxiliary subunit makes BK channels functionally relevant for circuit plasticity and behavior, with implications for understanding cerebellar ataxia.

Genomes are organized into chromatin loops through cohesin-mediated extrusion, with CTCF acting as a polar boundary element. As cohesin approaches CTCF at kilobase-per-second speeds, it must rapidly choose whether to stall or bypass. How CTCF encodes this probabilistic decision within a brief encounter window has remained unclear. Here we show that CTCF governs this probabilistic outcome by rapidly sampling a dynamic ensemble of conformations generated by spontaneous rearrangements of its DNA-binding zinc fingers. This ensemble is tuned by DNA sequence, CpG methylation, nearby nucleosomes, and the cohesin regulator PDS5A before cohesin engagement. Upon cohesin binding, PDS5A enhances loop-anchor mechanical stability, reinforcing orientation-dependent boundaries. These findings establish conformational ensemble tuning, rather than static occupancy, as a regulatory principle linking base pair-scale motions to megabase-scale genome organization.

Insufficient dietary fiber intake is strongly associated with gut microbiome dysfunction and an increased risk of noncommunicable diseases. Synergistic synbiotics, which pair defined microbial strains with their preferred carbohydrate substrates, offer a promising strategy to restore these functions. However, the rational design of such interventions remains challenging because of insufficient understanding of microbial fiber-degrading capacities and the host-relevant bioactivities of fermentation-derived metabolites. Here, we identify human colonic commensal Bacteroides intestinalis ( B . intestinalis ) as a key microbial mediator of dietary fiber-driven metabolic, immune, and neuronal benefits. We demonstrate that the synergistic interaction between B . intestinalis and its substrate, insoluble wheat arabinoxylan abundant in dietary fiber (inWAX), enhances the production of anti-diabetic and anti-steatotic bile acid species, anti-inflammatory and antioxidant phenolic compounds, and a spectrum of neuroactive compounds. These metabolic effects are accompanied by coordinated transcriptional remodeling in the colon and spleen implicating pathways governing circadian rhythm regulation, lipid metabolism, and immune defense. Importantly, these beneficial effects are preserved in conventionally raised mice with established high-fat diet-induced obesity, where B . intestinalis and inWAX improve glucose tolerance. Our findings uncover a mechanistic framework linking B . intestinalis -mediated fiber fermentation to gut-metabolism-immune crosstalk and establish a rational foundation for precision synbiotic design.

Spatial learning depends on hippocampal CA1 place cell representations, which form rapidly through behavioral timescale synaptic plasticity (BTSP). BTSP is driven by dendritic plateau potentials proposed to arise from the interaction of an excitatory target signal from entorhinal cortex layer 3 (EC3) and inhibitory feedback reflecting the current CA1 population state. However, the cellular source of this feedback has remained unknown. Using two-photon Ca ²⁺ imaging in mice during spatial learning, we found that dendrite-targeting oriens-lacunosum moleculare (OLM) interneurons increased their activity at behaviorally salient locations in a manner consistent with previously described environment-specific CA1 representations and EC3 target signals. Causal manipulations revealed that silencing a genetically defined subset of OLM interneurons late in learning enhanced BTSP and place field formation, whereas activating them early suppressed place field formation. These findings identify OLM interneurons as a key inhibitory feedback element regulating BTSP and the formation of hippocampal representations during learning.

Purpose: Intravesical interferon-alpha (IFNα) gene therapy has shown promise in treating BCG-unresponsive non-muscle invasive bladder cancer (NMIBC). Ongoing work in our lab aims to further improve its treatment efficacy by identifying resistance mechanisms and deploying targeted combination treatment strategies.

Experimental design: We performed end-tumor RNA-seq analysis of MB49 murine tumors treated with IFNα gene therapy, identifying the ErbB pathway as a resistance mechanism. We consequently hypothesized that a combination treatment involving an ErbB pathway blocker and IFNα could yield improved outcomes. MB49 cells were treated in vitro with lentiviral IFNα (LV-IFNα) gene therapy, with/without Afatinib, a pan-ErbB inhibitor, and cell viability and migration assays were performed. Next, in vivo studies were conducted in the syngeneic MB49 orthotopic murine bladder cancer model. The mice were randomized into 5 treatment groups (n=10 each): saline (Ctrl), LV-Ctrl, oral Afatinib monotherapy, intravesical LV-IFNα monotherapy, and the experimental intravesical LV-IFNα + oral Afatinib combination therapy. Overall survival (OS) and drug toxicity were assessed.

Results: Combination therapy significantly reduced MB49 cell viability in vitro compared to all other treatment conditions (mean relative ATPase activity at 72 h for the combination treatment was 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, respectively, p<0.001). This additive effect on cell viability appeared to be driven by a combination of early-cytostatic and late-cytolytic effects. The combination treatment also markedly inhibited cell migration (mean migrated cells/10x Boyden chamber assay at 36 h were: 92.3 for the combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, respectively, p<0.001). Finally, the in vivo studies demonstrated improved OS with combination therapy (median OS was 49 d in the combination group vs 15, 29, and 26 d in Ctrl, LV-IFNα, and Afatinib groups, respectively, Log-rank p<0.001). No mice in the combination therapy group died of drug toxicity.

Conclusions: Our preliminary findings suggest that the ErbB pathway may serve as a clinically significant resistance mechanism to intravesical IFNα gene therapy, and when targeted concurrently, may improve treatment efficacy.

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histones HctA and HctB. Here, we used an inducible CRISPR interference system to repress hctA, hctB, or both genes during development in Chlamydia trachomatis. Surprisingly, repression of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, when progeny EBs generated under histone-repressing conditions were used to initiate secondary infections in the absence of inducer, histone deficiency during EB maturation profoundly impaired fitness in the next infection cycle. Secondary cultures initiated with HctA-deficient EBs exhibited a delayed onset of genome replication, consistent with inefficient primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed genome replication and persistently reduced genome accumulation, indicative of defects in RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal a transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles. IMPORTANCE: Chlamydial histones HctA and HctB are unusual among bacterial chromatin-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on genome condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional chromatin factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

Copper is an essential micronutrient required by enzymes that catalyze oxygen-dependent reactions, but toxic in excess. Mutations in the ATP7A and ATP7B copper transporters cause neuropsychiatric symptoms and neurodegeneration by mechanisms that remain to be elucidated. We previously reported that the ATP7A biochemical interactome is enriched in Parkinson's disease (PD) and neurodegeneration associated proteins, yet the functional outcomes of these interactions are unknown. Using Drosophila , we tested genetic interactions between ATP7 mutants that alter copper levels and a subset of these PD and neurodegeneration causative genes and found sex differences with some candidate genes enhancing ATP7 deleterious phenotypes in both sexes, while others were sex specific. Most notably, we found that Lrrk2 (Lrrk), the most commonly mutated gene in familial forms of PD, protects against ATP7 dysfunction in epidermal epithelial cells with a stronger effect in males than females. However, in dopaminergic neurons Lrrk plays a role in intracellular copper induced toxicity in females but not males, supporting context dependent interactions between ATP7A and PD-associated genes to protect against disruptions in copper homeostasis.

Summary statement: We performed a genetic interaction screen to explore the relationship between copper homeostasis and Parkinson's disease and other neurodegeneration associated genes.

Online monitoring and quantification of neural signals has tremendous value both for neurofeedback experiments and for brain-computer interfaces. Unfortunately, established methods of online monitoring primarily involve the use of thresholded neural activity rather than sorted single-neuron spikes. The recent introduction of large-scale, high-density electrophysiology has enabled the recording of activity from hundreds of neurons simultaneously in both model organisms and human participants. This development highlights the need for a robust and easily implementable system for sorting spikes during data collection for live analyses of neuronal signals. Here, we describe a system for live sorting of neuronal activity (LSS) based on the widely used Kilosort platform. The LSS workflow utilizes an initial period of recorded neural data to identify waveform templates using Kilosort. LSS then interfaces with the SpikeGLX API to retrieve small batches (e.g. 50 ms) of data and for processing online. We measured the similarity of single-neuron activity sorted live by LSS to that sorted offline in neurophysiological recordings from macaque visual cortex using Neuropixels probes. We show that LSS closely replicates the post-stimulus time histograms and visual response tuning curves of single-neurons obtained using offline sorting. Furthermore, we show that decoding neural signals online with LSS consistently outperforms online decoding of thresholded activity, and that LSS can achieve the same performance as that obtained with offline sorting.