Biology subjects

Shi, Y.

Publications and source records attributed to Shi, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Genomic and Transcriptomic Landscapes of MEN1-Wild-Type Low-Grade Metastatic Pancreatic NETs Uncover Key Oncogenic Drivers and Targetable Pathways

Sporadic pancreatic neuroendocrine tumors (pNETs) with wild type MEN1 represent a major yet largely ignored subset whose biology and metastatic potential remain poorly understood. Because metastasis can occur despite low histologic grade and modest mutational burden, we hypothesized that metastatic competence in MEN1-wild-type pNETs reflects quantitative reinforcement of shared oncogenic pathways rather than distinct mutational processes. We profiled 75 primary low-grade pNETs by whole-exome and RNA sequencing, including 25 percent with lymph node and/or liver metastasis, and integrated genomic and transcriptomic data to connect pathway lesions with expression state. Metastatic tumors showed a slight increase in mutation frequency but conserved base-substitution spectra relative to non-metastatic cases, and adverse clinicopathologic features were enriched in Grade 2 disease. Aggregating alterations to pathways revealed broad convergence on canonical networks, with transcriptomic analyses demonstrating cohort-wide enrichment of Calcium, WNT, and KRAS/PI3K-AKT programs in metastasis. Intersection of significantly mutated genes with differentially expressed genes identified a focused 29-gene overlap, including RYR1 and ZNF273, that marks these convergent axes and distinguishes metastatic from non-metastatic tumors. Gene set enrichment confirmed preferential activation of Calcium, WNT, and PI3K-AKT signaling in metastatic tumors, consistent with a network-intensity model of progression. Finally, upstream-regulator analysis (iPathwayGuide) and gene-centric perturbation mapping (Gene2Drug) nominated candidate targeted and repurposable agents predicted to reverse the metastatic expression phenotype and flagged drugs unlikely to provide benefit, yielding a prioritized, testable therapeutic shortlist which includes fasudil and spaglumic acid. Convergent, domain-specific mutational patterns in highly mutated genes such as ZNF273 and CLCA1 define a molecular signature that could stratify metastatic risk in low-grade pNETs. Collectively, our data reframe metastasis in MEN1-wild-type low-grade pNETs as a property of pathway state rather than mutation quantity and provide a translational blueprint for biomarker-guided therapy development focused on Calcium, WNT, and KRAS/PI3K hubs.

cancer biology↗

bioRxiv2025

Oncometabolite fumarate impairs ATR-CHK1 signaling by succinating RPA1 in Fumarate Hydratase-deficient renal cell carcinoma cells

Abnormal accumulation of oncometabolite fumarate drives susceptibility in fumarate hydratase-deficient renal cell carcinoma (FH-dRCC), but the precise mechanisms remain not fully understood. In this study, we demonstrate that high fumarate levels impair activation of ATR-CHK1 signaling in response to replication stress and DNA damage. Mechanistically, fumarate modifies RPA1, an essential factor for ATR-CHK1 activation through succination, a post-translational modification. Succination of RPA1 occurs mainly at cysteine residues 481 and 486, which reduces its binding affinity for single-stranded DNA (ssDNA). RPA1 succination leads to deficient recruitment of TOPBP1 to ssDNA, resulting in attenuated CHK1 activation and defective cell cycle arrest in response to DNA damage. Succinated RPA1 compromises homologous recombination-mediated DNA repair. Our findings establish that fumarate-induced succination of RPA1 impairs DNA repair and cell cycle control, promoting genomic instability in FH-dRCC. This work reveals a novel mechanism by which oncometabolites contribute to genomic instability.

cancer biology↗

bioRxiv2025

A functionally selected Acinetobacter sp. phosphoethanolamine transferase gene from the goose fecal microbiome confers colistin resistance in E. coli

Polymyxins are last-resort antibiotics for infections caused by multidrug resistant Gram-negative bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. This makes the rise of bacteria exhibiting polymyxin E (colistin) resistance, largely through modification of lipid A moieties, concerning and suggests that it is important to document potential sources of the corresponding resistance genes. This study searched for potential emerging colistin-resistance genes from the environment by investigating a previously performed functional metagenomic selection for colistin resistance of a goose fecal microbiome. We found that the selection captured Acinetobacter sp. DNA fragments which all contained eptA genes. We confirmed their ability to confer significant colistin resistance in E. coli via modification of lipid A in the outer membrane. Furthermore, we found evidence for mobilization of closely related eptA genes in Acinetobacter strains, marking them as potential mcr genes or their precursors. This study highlights the goose fecal microbiome as a potential source for colistin resistance in the environment.

microbiology↗

bioRxiv2025

Ethylene signal-driven plant-multitrophic synergy boosts crop performance

Efficient nutrient use in agriculture depends on the dynamic interplay between plant roots, soil, and microbial communities. The root-rhizosphere interface is central to nutrient uptake and serves as a key hub for interactions with beneficial microbes. Arbuscular mycorrhizal (AM) fungi, positioned at the nexus between plant roots and soil microbiota, play a critical role in enhancing crop performance under nutrient-limited conditions. In this study, we dissected the genetic and molecular basis of AM fungi-induced lateral root development in maize (Zea mays), focusing on the role of ethylene-responsive transcription factors (ERFs). We identified ERF genes as essential regulators of pericycle cell division, acting downstream of ethylene biosynthesis genes (ACS6 and ACS7) and AM fungal signaling. Our findings reveal that AM fungi promote lateral root initiation by activating ERF expression and reprogramming flavonoid metabolism, particularly reducing the accumulation of flavonols such as kaempferol and quercetin, which otherwise inhibit root development when over-accumulated. Furthermore, we demonstrated that Massilia, a beneficial rhizobacterium, synergizes with AM fungi to enhance lateral root formation by colonizing fungal hyphae, degrading flavonoids, and contributing to auxin production. Together, our results uncover a tripartite signaling network linking ethylene signaling, flavonoid-mediated microbial recruitment, and AM symbiosis. This study highlights ERFs as central integrators of plant-microbe interactions and provides a molecular framework for engineering root architecture and microbiome assembly to improve nutrient acquisition and support sustainable crop productivity.

plant biology↗

bioRxiv2025

Co-targeting KRAS and Exportin1 as an effective therapeutic strategy for KRASG12D mutant pancreatic ductal adenocarcinoma

BackgroundSeveral KRASG12D inhibitors (KRASG12Di) are under clinical evaluation for pancreatic ductal adenocarcinoma (PDAC). However, as seen with other first generation KRAS inhibitors, resistance may limit their long-term efficacy, necessitating combination strategies to enhance therapeutic outcomes. Exportin 1 (XPO1), a nuclear transport protein overexpressed in PDAC, represents a therapeutic vulnerability in KRAS-mutant cancers. Here, we demonstrate that the second-generation XPO1 inhibitor Eltanexor synergizes with MRTX1133 to enhance its efficacy in multiple PDAC models. MethodsWe generated KRASG12Di-resistant PDAC cells and assessed their response to Eltanexor. The antiproliferative effects of MRTX1133 and Eltanexor combinations were evaluated in 2D and 3D in vitro PDAC models. The in vivo efficacy of the combination was tested in KRASG12D-mutant human and murine PDAC xenograft and allograft models. ResultsEltanexor sensitized MRTX1133-resistant PDAC cells to growth inhibition. In both 2D and 3D culture models, the combination of Eltanexor and MRTX1133 significantly reduced cell viability. Mechanistically, the combination treatment suppressed key KRAS downstream signaling molecules, including p-ERK, mTOR, p-4EBP1, DUSP6, and cyclin D1. Kinome analysis further revealed reduced MAPK-related kinase activity. Combining subtherapeutic doses of Eltanexor and MRTX1133 resulted in significant tumor regression and prolonged survival in PDAC xenograft and immunocompetent orthotopic allograft models. Moreover, maintenance therapy with Eltanexor prevented tumor relapse, yielding a durable antitumor response. ConclusionThis study demonstrates that Eltanexor overcomes resistance to MRTX1133 and enhances its efficacy in PDAC. The combination regimen may provide a durable therapeutic response while reducing the required dose of KRASG12D inhibitors, potentially delaying resistance and improving patient outcomes. Statement of Translational RelevancePDAC remains one of the deadliest malignancies, with limited effective therapies and dismal survival rates. The emergence of KRASG12D-selective inhibitors, such as MRTX1133, marks a critical advance for nearly 40% of PDAC patients harboring this oncogenic driver. However, inevitable emergence of adaptive or acquired resistance to KRAS inhibitors remains a major barrier to achieving durable clinical benefit. This study uncovers XPO1 inhibition as a rational and synergistic strategy to augment the antitumor efficacy of MRTX1133. By enhancing KRASG12D inhibitor activity and potentially reducing the required therapeutic dose, this combination approach offers a novel means to delay or overcome resistance. These findings provide a strong preclinical rationale for clinical trials evaluating KRAS inhibitors in combination with XPO1 inhibitors and may significantly improve outcomes for a substantial subset of PDAC patients who currently lack effective targeted treatment options.

cancer biology↗

bioRxiv2025

The Human-In-the-Loop Drug Design Framework with Equivariant Rectified Flow

Advancements in AI-based drug design often face obstacles due to incomplete datasets, hindering progress to clinical trials. Human experts bring invaluable expertise and nuanced contextual understanding to drug design. There are two main difficulties in integrating human knowledge into the drug development process. First, human annotations are costly, and traditional machine learning algorithms require a large number of samples to be effective. Second, human experts are unable to accurately describe their expertise using natural language, nor can they precisely specify what kind of molecule is needed before seeing the generated molecules. To address these problems, we propose a new platform, called HIL-DD, for experts to infuse their experience by selecting molecules generated by AI that meet their criteria, or discarding those that do not. The core generative technology utilizes an Equivariant Rectified Flow Model (ERFM), which offers faster generation speeds than conventional diffusion models, enabling efficient human-AI collaboration. More importantly, we provide a user-friendly interface to ensure smooth and effective collaboration between human experts and AI systems. Rigorous experiments demonstrate that our system can produce 3D molecules that align with expert expectations in minimal interactive sessions. These generated molecules maintain drug-like qualities comparable to those created by current state-of-the-art models.

bioinformatics↗

bioRxiv2025

Immune-Derived THBS1-CD47 Axis Induces Cellular Senescence and Suppresses Osteogenesis in Diabetic Periosteum

Type 2 diabetes mellitus (T2DM) weakens bone repair and increases fracture risk, but how the periosteal environment contributes to this problem is not fully understood. We found that diabetic mice showed thinning of the periosteum, reduced bone mass, and poor function of bone-forming cells. Using single-cell RNA sequencing, we discovered that immune cells and bone-lineage cells interact more strongly in diabetes through the THBS1-CD47 pathway. THBS1 was mainly produced by macrophages, while its receptor CD47 was abundant in osteogenic cells. Laboratory experiments showed that THBS1 blocked bone gene activity, increased inflammation and cell death, and damaged mitochondria, leading to higher oxidative stress. Knocking down CD47 reversed these effects, restoring bone cell activity and energy balance. In diabetic fracture models, blocking THBS1 improved callus formation and bone healing. These findings identify THBS1-CD47 as a key driver of periosteal dysfunction in T2DM and highlight it as a potential target to improve skeletal repair in diabetic patients.

cell biology↗

bioRxiv2025

Phospho 394Y LCK flow-cytometry readout predicts dasatinib sensitivity in paediatric T-cell acute lymphoblastic leukaemia

Children with T-cell acute lymphoblastic leukaemia (T-ALL) who relapse or fail induction have poor outcomes. A subset of cases shows glucocorticoid resistance reversible by dasatinib through inhibition of LCK-dependent signalling. To identify a practical biomarker of dasatinib response, we compared in-vitro drug sensitivity with basal phosphorylation of pre-TCR pathway proteins in 28 paediatric T-ALL patient-derived xenografts (PDX). Phospho-flow cytometry quantified LCK (pY394), ZAP70 (pY319) and CD3{zeta} (pY142), normalised to internal controls. Dasatinib IC50 values correlated significantly with pLCK and pZAP70, with pLCK providing the best single-marker performance across clinically relevant thresholds. Logistic, ROC and precision-recall analyses confirmed that pLCK alone achieved excellent classification (AUC [≥] 0.9), while multivariate models added minimal predictive value. Model selection using LASSO and Bayesian Information Criterion further supported pLCK as the dominant predictor. These findings establish pLCK as a robust, scalable biomarker of dasatinib sensitivity suitable for diagnostic integration. A multicentre international validation programme is underway to harmonise pLCK assay protocols, expand testing across biobanks, and assess clinical feasibility in newly diagnosed and relapsed patients within the ALLTogether and HEM-iSMART trials respectively.

cancer biology↗

bioRxiv2025

Targeting the host factor HGS-viral membrane protein interaction in coronavirus infection

While current antivirals primarily target viral proteins, host-directed strategies remain underexplored. Here, we performed a genome-wide CRISPRi screening to identify the host protein, Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS), as essential for the pan-coronaviruses infection both in vitro and in vivo. Mechanistically, HGS directly interacts with the viral membrane (M) protein, facilitating its trafficking to the ER-Golgi intermediate compartment (ERGIC) for virion assembly. Conversely, HGS deficiency caused M retention in the ER, blocking assembly. Leveraging this interaction, we designed M-derived peptides and screened over 5,000 FDA-approved drugs, identifying riboflavin tetrabutyrate (RTB). Both the peptides and RTB bind HGS and disrupt its interaction with the M protein, leading to M retention in the ER and subsequent blockade of virion assembly. These agents demonstrated broad anti-pan-coronavirus activity in vitro and in vivo. Collectively, our findings establish HGS as a druggable host target and identify RTB as a promising broad-spectrum antiviral candidate.

microbiology↗

bioRxiv2025

Stage-resolved Spatial Multi-omics Reveals Myeloid Niches in Human Atherosclerotic Plaques

BackgroundAtherosclerosis, a leading cause of heart attack and stroke, involves intricate immune cell dynamics within arterial plaques, yet their spatial organization and functional roles remain elusive. MethodsWe combined Visium HD spatial transcriptomics, imaging mass cytometry, and single-cell RNA-seq across fatty-streak, advanced, and restenotic plaques to map myeloid architectures and relate them to lesion geography and extracellular matrix features. Slingshot trajectory analysis resolved macrophages differentiation path. In vitro, we tested microenvironmental and lipid cues separately: fibronectin (FN) exposure during PMA-driven THP-1 differentiation and oxidized LDL (oxLDL)-induced foam cell formation in murine bone-marrow derived macrophages (BMDMs). ResultsOur analysis identified seven macrophage subsets and two neutrophil populations with distinct spatial distribution and functional roles. In early lesions, neutrophils expressing MMP9, MPO, p47phox, TGF-{beta}1 and arachidonate 5-lipoxygenase (ALOX5), aligned with proteolysis, inflammatory processes, and endothelial-mesenchymal transition features. In advanced plaques, macrophage subsets exhibit specialized functions: Ki67+ proliferative macrophages localized near necrotic cores, sustaining local population; SPP1+ macrophages, enriched in lipid handling and tissue remodeling, are prone to apoptosis/ferroptosis, potentially promoting necrotic core expansion; and C3aR+ macrophages form antigen-presenting niches with elevated HLA-DR and CD74, engaging T cells possibly through CXCL12-CXCR4 signaling. Slingshot trajectories indicated progression from C3aR toward SPP1 remodeling states concentrated at fibronectin-rich rims. In vitro, FN increased MMP9 and TIMP1 in THP-1-derived macrophages, consistent with FN imprinting remodeling features characteristic of SPP1 macrophages in situ. Concurrently, oxLDL-treated BMDMs showed enhanced lipid-handling and remodeling modules consistent with the SPP1 program. ConclusionsThese findings define conserved myeloid niches and support a microenvironment-imprinting model that links ECM composition and lipid loading to macrophage state transitions, providing a framework for microenvironment-targeted therapies to stabilize plaques and mitigate cardiovascular risk.

pathology↗

bioRxiv2025

FlexRibbon: Joint Sequence and Structure Pretraining for Protein Modeling

AO_SCPLOWBSTRACTC_SCPLOWProtein foundation models have advanced rapidly, with most approaches falling into two dominant paradigms. Sequence-based language models (e.g., ESM-2) capture sequence semantics at scale, and a number of recent works incorporate structural signals into sequence encoders. MSA-based predictors (e.g., AlphaFold 2/3) achieve accurate folding by exploiting evolutionary couplings, but their reliance on homologous sequences makes them less reliable in highly mutated or alignment-sparse regimes. We present FlexRibbon{ddagger}, a pretrained protein model that jointly learns from amino acid sequences and three-dimensional structures. Our pretraining strategy combines masked language modeling with diffusion-based denoising, enabling bidirectional sequence-structure learning without requiring MSAs. Trained on both experimentally resolved structures and AlphaFold 2 predictions, FlexRibbon captures global folds as well as flexible conformations critical for biological function. Evaluated across diverse tasks spanning interface design, intermolecular interaction prediction, and protein function prediction, FlexRibbon establishes new state-of-the-art performance on 12 different tasks, with particularly strong gains in mutation-rich settings where MSA-based methods often struggle.

molecular biology↗

bioRxiv2025

Multiomic profiling of a unique in-transit melanoma cohort identifies melanoma differentiation as predictor of tumor progression and therapy response

Melanoma patients with in-transit metastasis (ITM), a stage of disease where melanoma has metastasized to sites in between the primary lesion and draining lymph node, vary significantly in their clinical outcomes, but the biology driving differential outcomes in ITM is poorly understood. To elucidate the mechanisms of differential outcomes, we utilized multimodal molecular profiling (WES, RNA-seq, highly multiplexed immunofluorescence, spatial transcriptomics) in 1) evolutionary analysis of longitudinal tumor samples and 2) identifying prognostic tumor intrinsic and microenvironmental features in a unique cohort of patients with unresectable ITM. Among other findings, we observed a persistent dedifferentiated AXL/NGFR clonal lineage pre-existing and following immune checkpoint blockade in in-transit and distant metastases. Concordantly, we found that low pigmentation and high T cell exhaustion signatures were independently associated with distant progression. Our findings highlight tumor cell state and immune dysfunction as key predictors and potential biomarkers of metastatic risk in ITM. STATEMENT OF SIGNIFICANCEWhat drives distant progression in melanoma is unclear. Analyzing tumor and immune features in a rare in-transit melanoma patient cohort, we identify biological signals highlighting how immune and tumor states observable in pre-distant metastasis melanomas shape long-term outcomes, and nominate potential prognostic biomarkers.

cancer biology↗

bioRxiv2025

Vascular and Lymphatic Dysregulation via Non-EndoMT Col2a1 Signaling in Bisphosphonate-Related Osteonecrosis of the Jaw

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is one of the most severe complications of antiresorptive therapy. Although impaired angiogenesis is commonly observed in BRONJ, the underlying vascular mechanisms remain poorly understood, impeding the development of effective treatments. In this study, we integrated whole-organ tissue-clearing imaging, single-cell transcriptomics, and proteomics to construct a multimodal atlas of the mandibular microenvironment in both mouse and human BRONJ. Our findings revealed gross vascular and lymphatic rarefaction in BRONJ, along with 4 endothelial subtypes uniformly exhibiting aberrant upregulation of Col2a1. Moreover, fibroblasts and macrophages emerged as key endothelial interactors, collectively underscoring dysregulation of the endothelium-matrix-immune axis. Unlike homeostatic Dmp1/Tfap2a fibroblasts and Stab1 macrophages, BRONJ lesions featured pathological Lrrc15/Chad fibroblasts and Il6 macrophages, which promote ectopic chondrogenesis and inflammation. Mechanistically, BRONJ activated the COL2A1-CD44 axis (EC-to-fibroblast/macrophage signaling) and COL2A1-SDC4 axis (EC-to-fibroblast signaling), whereas lineage tracing excluded endothelial-to-mesenchymal transition (EndoMT), implicating extracellular matrix remodeling as the driver of dysfunction. Cross-species validation in human mandibles further confirmed vascular-lymphatic dysregulation, inflammatory activation, and aberrant chondrogenesis. Overall, this study establishes vascular and lymphatic dysfunction as a central pathological hallmark of BRONJ, driving matrix remodeling dysregulation and immune-inflammatory imbalance, and identifies the COL2A1-CD44/SDC4 axis as a potential therapeutic target for BRONJ.

cell biology↗

bioRxiv2025

Phosphoantigen-Driven Dissociation of Butyrophilin Oligomers Activates γδ T Cells

{gamma}{delta} T cells represent a promising avenue for cancer immunotherapy. The V{gamma}9V{delta}2 T cell receptor (TCR), expressed by the predominant subset of {gamma}{delta} T cells, responds to phosphoantigen (pAg)-engaged butyrophilins (BTNs) on various cancer cells. However, the molecular mechanism underlying pAg-mediated activation of V{gamma}9V{delta}2 TCRs remains a subject of debate. Here, we employed an integrative approach to elucidate the mechanism of pAg reactivity in V{gamma}9V{delta}2 T cells. Our results demonstrate that BTNs form higher-order oligomers in the absence of pAg. Upon pAg binding, these oligomers dissociate into tetramers, enabling V{gamma}9V{delta}2 TCR engagement. This pAg-induced dissociation of higher-order BTN complexes is critical for pAg-mediated activation of {gamma}{delta} T cells. Our findings reveal a mechanism of BTN dissociation-driven pAg sensing, providing valuable insight for future immunotherapeutic strategies.

immunology↗

bioRxiv2025

The Genetic Immune Basis of Gout: Identification, Functional Characterization, and Therapeutic Implications

Gout, the most common inflammatory arthritis, is still managed mainly as a metabolic disorder, with treatment centered around urate lowering rather than blocking the immune response that drives gout flares. This emphasis reflects a historical view of gout as a disease of excess and diet, reinforced by genome-wide association studies (GWAS) that consistently detect urate transport and metabolism loci, while immune-related loci have failed to reach genome-wide significance. Growing clinical and experimental evidence, however, points to a central role for immune pathways in gout pathogenesis. We set out isolate the immune genetic component of gout. Using a conjunctional false discovery rate (conjFDR) framework, we integrated gout GWAS data with those from eight immune-mediated disorders rheumatoid arthritis, Crohns disease, inflammatory bowel disease, psoriasis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, and systemic lupus erythematosus. We observed robust pleiotropic enrichment across all comparisons and mapped the resulting loci through FUMA to 85 unique credible genes broadly distributed across the genome, with no classical urate transporters present. Additionally, we identified 16 novel genes for gout, many of which are of obvious immune nature. We performed GO and KEGG enrichment analyses, which at a stringent q-value threshold identified adaptive and innate immune pathways, including T and B cell receptor signaling, antigen processing and presentation, NF-{kappa}B signaling, and Jak-STAT signaling. At a nominal p-value threshold, we uncovered additional cytokine-driven processes such as IL6 and IL7 signaling. We confirmed disease relevance with DisGeNET and established causality for 14 genes through Mendelian randomization, including IL1RN, MAP3K11, and SH2B3 genes with existing pharmacological inhibitors. We are the first to genetically isolate the genetic immune component of gout. Our findings show that these immune pathways can be specifically targeted, and immune medication should be incorporated into therapeutic strategies to complement urate-lowering approaches.

immunology↗

bioRxiv2025

DNA Data Storage Architecture via Ligation of Dynamic DNA Bytes

The explosive growth of digital data is overwhelming conventional storage media, creating an urgent need for more efficient solutions. DNA offers immense potential for digital data storage, yet most systems remain static and archival. Here, we present a modular DNA storage architecture based on dynamic DNA bytes (DynaBytes)-- pre-fabricated DNA segments that can be ligated into reconfigurable information units. Utilizing core, functional and control DynaBytes, we stored 210,776 bits (26,347 bytes) of digital information organized within a file-system, and demonstrated CRUD (Create-Read-Update-Delete)-like operations, hierarchical access and nanopore-based realtime retrieval. Robust data recovery was achieved under [~]100x error-prone sequencing using streamlined error correction and fuzzy decoding. By relying on in vitro ligation of standardized components, DynaBytes reduces cost, scales efficiently, and allows interactive, rewritable storage. These features advance DNA storage beyond passive archiving toward a reconfigurable framework, opening new possibilities for dynamic, practical and large-scale DNA-based data systems.

synthetic biology↗

bioRxiv2025

Small Single-Stranded DNA Structure Prediction

Small single-stranded DNAs (ssDNAs) shorter than 100 bases, including antisense oligonucleotides and aptamers, have been extensively studied as promising therapeutics. Their three-dimensional (3D) structures can guide development and enhance clinical potential, but obtaining these structures is often costly and time-consuming. Only a limited number of small ssDNAs have their 3D structures experimentally determined. Therefore, there is a significant need to develop accurate computational tools to predict the 3D structures of small ssDNAs. In this work, we analyze two current strategies for ssDNA structure prediction: a traditional multistep approach and the AlphaFold 3 (AF3) strategy. We compare these strategies using a dataset of 36 ssDNAs, representing 7 unique structural motifs. Our results show that AF3 can be more efficient and accurate than the traditional strategy for predicting ssDNA structures, achieving a success rate of 56% (20 out of 36 cases). However, AF3 faces some challenges when predicting long ssDNA sequences or complex motifs, such as G-quadruplexes (G4) and junctions. Interestingly, molecular dynamics (MD) simulations can significantly improve the predicted structures generated by AF3. These findings offer valuable insights into the latest ssDNA structure prediction techniques and provide a practical guide for sampling ssDNA structures with AF3 and MD.

biochemistry↗

bioRxiv2025

ATF4 alleviates doxorubicin-induced cardiomyopathy through H2S-mediated antioxidation

BackgroundDoxorubicin (DOX) remains a cornerstone in the treatment of various cancers. However, its clinical utilization is significantly hampered by dose-dependent cardiotoxicity. The generation of reactive oxygen species (ROS) constitutes the central component of the pathogenesis of DOX-induced cardiotoxicity. Activating transcription factor 4 (ATF4) has been demonstrated to exert a cardioprotective effect and augment cardiac antioxidative capacity in settings of heart failure. However, the role of ATF4 in DOX-induced cardiomyopathy (DIC) remains unknown. MethodsTo explore the role of ATF4 in DOX-induced cardiomyopathy, cardiac-specific ATF4 conditional heterozygous mice and AAV9 mediated ATF4 overexpression mouse models were utilized. Cardiac function was assessed by echocardiography. The upstream regulator and downstream mediator of ATF4 were evaluated using RNA-seq analysis and further verified using ChIP assay and luciferase reporter assay. ResultsWe found a substantial decrease in ATF4 expression levels in the heart of DIC mice. ATF4+/- mice exhibited a higher degree of susceptibility to DOX-induced cardiotoxicity in comparison with ATF4flox/flox mice, as evidenced by the manifestation of more severe cardiac dysfunction and a significantly earlier mortality rate. In contrast, cardiacc-specific overexpression ATF4 by AAV9 confers robust cardioprotection against DOX-induced cardiomyopathy. Mechanistically, we identified the upstream regulator of ATF4 as KLF16, which was significantly suppressed during DOX treatment. Further, the decrease of ATF4 led to a reduction in cystathionine {gamma}-lyase (CSE) transcription and hydrogen sulfide (H2S) production in the context of DOX-induced cardiotoxicity. ChIP and luciferase reporter assays revealed that ATF4 functioned as the transcription factor of the CSE gene, which is a key enzyme in the synthesis of H2S to counteract oxidative stress. Consistently, ROS scavengers or H2S donors was shown to mitigate the consequences of ATF4 deficiency. In contrast, the ectopic expression of ATF4 mitigated oxidative stress and apoptosis in DOX-induced cardiotoxicity, both in vivo and in vitro. ConclusionsOur study revealed a novel function of ATF4 in counteracting oxidative stress in DOX cardiotoxicity by promoting the transcription of CSE. ATF4 may represent a promising therapeutic target for the treatment of DOX-induced cardiomyopathy.

pathology↗