bioRxiv : the preprint server for biology最新文献

The ovary is one of the first organs to lose functionality with age. We found that aging of the Drosophila ovary is characterized by an accumulation of phenotypes in the somatic compartment, including failure of the follicle cells to encapsulate germ-cell cysts, an extended S phase, and increased DNA damage. In aged ovaries, follicle encapsulation defects are associated with the lack of a germ-cell cyst checkpoint in early oogenesis. Single-cell RNA sequencing revealed that, across all cell types in the ovary, cells in the follicle lineage have the highest number of differentially expressed genes. Overexpression of Atg8a, a key autophagy machinery gene homologous to mammalian LC3, specifically in follicle cells prevents age-associated decline in the follicle epithelium and loss of reproductive capacity. Collectively, these findings demonstrate that genetic manipulation of a small population of ovarian somatic cells is sufficient to improve both cell-autonomous and non-autonomous features of reproductive aging.

Phenotypic plasticity is a prominent cancer feature that contributes to metastatic potential and resistance to therapy across multiple cancer types. Cancer cell state transitions have been attributed to transcriptional programs, such as the AP1/TEAD-regulated gene network driving the mesenchymal-like (MES) phenotype. In addition, during dissemination, tumor cells are subjected to variable loads of physical mechanical pressure and constriction across transited tissue, which are thought to impact nuclear molecular crowding. How the interplay between mechanical pressure, global 3D nuclear architecture and transcriptional programs contributes to MES identity and metastatic adaptation remains unclear. Using cutaneous melanoma as a model for early dissemination, we integrate in vitro and in vivo epigenomic profiling with nanoscale imaging of cell lines and patient samples to investigate chromatin organization features underlying the MES phenotype. We find that in MES cells, CTCF is relocated from domain boundaries to regulatory regions of EMT-like genes, leading to reduced insulation, extended topological associated domains (TADs) and increased inter-domain contacts, and de novo formation of chromatin hubs. This conformational rewiring, along with loss of heterochromatin, supports nuclear deformability during invasion and dissemination. Conversely, physical constriction of melanocytic cells induces MES-like chromatin features, including CTCF repositioning and heterochromatin loss, and promotes metastasis in vivo. Similarly, pharmacological inhibition of the heterochromatin mark H3K9me3 triggers MES characteristics and increases invasiveness. These results demonstrate that metastatic competency involves both epigenetic and structural nuclear reprogramming, enabling shifts in gene networks and physical adaptability. Our findings reveal mechanistic links between nuclear architecture and aggressive tumor behavior, identifying potential biomarkers and therapeutic targets to intercept metastatic progression.

Virtual screening for small-molecule binders is often limited by false positives from approximate scoring functions and rigid-receptor assumptions. These can be addressed downstream through accurate but expensive free energy calculations. At the same time, recent artificial-intelligence-based co-folding methods have been proposed that claim to achieve accuracy of free energy methods at much lower cost, but these have not yet delivered consistent improvements in early enrichment and can be confounded by memorization. Here we address this gap by introducing c(t)-based metadynamics (CTMD), a physics-based, high-throughput hit-triaging protocol tailored for early enrichment. CTMD uses the nonequilibrium reversible-work estimator c(t) introduced by Tiwary and Parrinello (Journal of Physical Chemistry B, 2015 119 736), computed from a small number of short, independent well-tempered metadynamics trajectories, to rank binding stability without requiring converged binding free energies. Across diverse targets and chemotypes, CTMD provides robust early enrichment while remaining fast, transferable with minimal parameter tuning, and resistant to memorization-driven artifacts - underscoring both an immediately deployable physics-based alternative for screening. For these systems we show how co-folding, particularly Boltz-2, achieves enrichment directly proportional to similarity with training set, and more worrying, even in the presence of significant modifications to active site. Given its simplicity of implementation, CTMD should thus be an "embarassingly" open-source, early enrichment method available for use by the broad pharma and academic community that sits right between approximate but fast docking or AI based co-folding methods, and more expensive but accurate free energy calculations, expected to lead to saving significant financial and human capital in drug discovery campaigns.

Background: Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as less than 50% of patients with melanoma achieve long-term response to immune checkpoint inhibitor therapy. One mechanism of acquired resistance involves somatic mutations, such as loss of beta-2 microglobulin (B2m), that enable tumor cells to evade T cell-mediated killing.

Methods: This study used single-cell RNA-seq, flow cytometry, and ex vivo functional assays to characterize tumor-infiltrating immune cells in antigen presentation-deficient tumors. Tumor-bearing mice were treated with anti-PD-1 or CD40 agonist antibodies and cell depletion or cytokine blocking antibodies to define mechanisms of action. Analysis of published human RNA-seq datasets was performed to dissect the contributions of inflammatory monocytes to patient outcomes.

Results: We found an increase in immunosuppressive macrophages in B2m-null tumors. We hypothesized that repolarizing myeloid cells may restore control of tumor growth. Treatment with CD40 agonist antibody, which promotes differentiation of monocytes and macrophages towards a proinflammatory phenotype, reduced tumor growth and improved survival in B2m-null melanoma and colorectal cancer models. Unexpectedly, both CD8+ T cells and NK cells, but not CD4+ T cells, were required for the efficacy of CD40 agonist, even though CD8+ T cells cannot directly recognize antigen presentation-deficient tumor cells. Instead, these lymphocytes control tumor growth via secretion of IFNγ, as depletion of IFNγ inhibited the therapeutic effect of CD40 agonist. IFNγ receptor (Ifngr1) expression was required on host cells, not tumor cells, for CD40 agonist-mediated tumor control. Single-cell analysis identified a distinct population of inflammatory monocytes that were enriched for an IFNγ response signature in CD40 agonist-treated tumors, suggesting that these cells may be important for tumor control. Analysis of human bulk and single-cell RNA-seq datasets demonstrated that an inflammatory monocyte signature derived from our data was associated with improved patient outcomes and response to immune checkpoint inhibitors.

Conclusions: These data demonstrate that CD8+ T cells contribute to tumor control even in the absence of direct antigen presentation by tumor cells. More broadly, our work suggests that strategies to activate the effector functions of inflammatory monocytes may limit tumor growth and overcome acquired resistance to immune checkpoint inhibitors.

Solid stress shapes tumor growth, invasion, and therapeutic response, yet its physical origin and clinical relevance remain unclear. Here, we develop a mechano-electro-osmotic model integrating metabolic gradients, ion transport, and cellular mechanics to explain residual solid stress emergence in tumor spheroids, common models of solid tumors. We show that solid stress arises predominantly from osmotic cell swelling driven by metabolic deprivation and ion accumulation, rather than proliferation. This mechanism generates a characteristic stress architecture: isotropic compression in the hypoxic core balanced by peripheral tangential tension, causing pronounced cell and nuclear deformation. The resulting nuclear strain provides a mechanical basis for DNA damage and genomic instability implicated in disease progression and treatment resistance. We validate these predictions in breast cancer using MDA-MB-231 spheroids and patient-derived ductal carcinoma in situ lesions, and corroborate them across published spheroid models and in vivo and ex vivo tumors spanning additional cancer types. Our findings link tumor metabolism to clinically relevant mechanical stresses, suggesting opportunities to target osmotic and metabolic pathways to mitigate solid stress and improve therapeutic outcomes.

Motivation: Large-scale genomic biobanks contain thousands of second-degree relatives with missing pedigree metadata. Accurately distinguishing half-sibling (HS) from niece/nephew-avuncular (N/A) pairs-both sharing approximately 25% of the genome-remains a significant challenge. Current SNP-based methods rely on Identical-By-Descent (IBD) segment counts and age differences, but substantial distributional overlap leads to high misclassification rates. There is a critical need for a scalable, genotype-only method that can resolve these "half-degree" ambiguities without requiring observed pedigrees or extensive relative information.

Results: We present a novel computational framework that achieves near-complete separation of HS and N/A pairs using only genotype data. Our approach utilizes across-chromosome phasing to derive haplotype-level sharing features that summarize how IBD is distributed across parental homologues. By modeling these features with a Gaussian mixture model (GMM), we demonstrate near-perfect classification accuracy (> 98%) in biobank-scale data. Furthermore, we show that these high-confidence relationship labels can serve as long-range phasing anchors, providing structural constraints that improve the accuracy of across-chromosome homologue assignment. This method provides a robust, scalable solution for pedigree reconstruction and the control of cryptic relatedness in large-scale genomic studies.

Copper chalcogenide nanocrystals (NCs) are promising candidates for biophotonic applications due to their tunable optical properties. Concrete methods to examine the relationship between their degradation and toxicity are necessary to enable development of nanoconstructs with reduced toxicity. This study compares the degradation and acute cytotoxicity of three compositions of micelle-coated copper chalcogenide NCs: the fluorescent semiconductor copper indium sulfide (CuInS ₂ ), and the plasmonic semiconductors copper sulfide (Cu _2-x S) and chalcopyrite copper iron sulfide (CuFeS ₂ ). We developed a quantitative degradation assay to assess ion release from these ultra-small nanocrystals, revealing that while all three particles biodegrade, CuInS ₂ and CuFeS ₂ undergo rapid degradation in artificial lysosomal fluid, leading to a burst release of indium and iron ions. In cellular toxicity assays, CuInS ₂ exhibited significantly higher acute cytotoxicity than Cu _2-x S and CuFeS ₂ , primarily due to indium-induced necrosis. To mitigate this toxicity, an alternative surface-binding polymer coating was introduced, effectively reducing both the degradation rate and cytotoxicity of CuInS ₂ . These findings highlight the influence of both nanocrystal composition and coating chemistry in moderating the acute cytotoxity of degradable nanocrystals, demonstrating that tuning of composition and degradation rate can be used to moderate nanoparticle toxicity.

Eukaryotic DNA is organized across multiple scales to support genome compaction, appropriate gene expression, and DNA recombination. A central player in these roles is the CCCTC binding factor (CTCF), which defines specific chromatin loop structures and insulates enhancer elements from promoters. Chromatin is organized in a distinct pattern around CTCF-bound sites, however, the role of this patterning remains unclear. Here, we report cryo-electron microscopy structures of reconstituted CTCF-nucleosome complexes, revealing that CTCF dimerization promotes the oligomerization of nucleosomes into defined higher-order assemblies involving specific histone-histone and CTCF-CTCF interactions. Notably, CTCF does not oligomerize efficiently on non-chromatinized DNA substrates. Disruption of CTCF-CTCF interaction interfaces in cells results in a marked decrease in chromatin looping and impairs cellular differentiation. These results indicate that chromatin structure at CTCF sites plays an important role in supporting higher-order interactions between distal regions of the genome and that these interactions are important for supporting cell-type-specific gene expression.

A small burn can render a large area of skin painfully tender. This widespread hyperalgesia protects injured tissue and is typically attributed to altered spinal cord mechanisms. Whether peripheral sensory afferents directly interact to contribute to hyperalgesia remains unclear. Using single-unit microneurography, we recorded cutaneous afferents before and after inducing localized TRPV1-mediated inflammatory flare. This triggered minutes-scale reweighting of transcriptomically defined TRPV1 ^- afferents, with divergent effects among myelinated (Aβ-range) classes: tactile receptors showed reduced responsiveness, whereas mechano-nociceptors underwent sensitization. These changes paralleled diminished tactile sensitivity and intensified mechanical pain. Recruitment of mechanically silent branches in Aβ-range mechano-nociceptors produced wide-field amplification of peripheral nociceptive signaling beyond the inflamed site. These findings suggest that rapid peripheral crosstalk from TRPV1 ⁺ afferents reprograms TRPV1 ^- Aβ-afferents and drives human hyperalgesia.

Shwachman-Diamond syndrome (SDS) is a ribosomopathy characterized by neutropenia, pancreatic insufficiency, skeletal defects, and predisposition to leukemia. Most cases result from biallelic SBDS mutations that impairing 80S ribosome and polysome assembly. In yeast lacking SDO1 (the SBDS ortholog), growth slows dramatically and the p38 ortholog Hog1 signaling is elevated by multiple types of stress. SBDS-deficient HeLa cells exhibited reduced proliferation and slowed cell cycling. The p38 kinase was constitutively activated in SBDS mutants and SDS patient-derived blood cells. Because ZAKα detects ribosome dysfunction, its activation links ribosomal defects to stress kinase pathways in SDS. Suppressing p38α or its upstream activator ZAKα restored cell growth and reduced stress signaling. These findings reveal an evolutionarily conserved-independent mechanism via p38 drives SDS pathophysiology and identifies stress kinases as potential therapeutic targets for ribosomal dysfunction.