Abstract Designing microbial communities to generate target products is crucial for biotechnology, agriculture, and disease treatment. However, rationally designing such communities from large seed pools has become a major challenge, as the rapidly expanding number of complete microbial genomes greatly expands the search space and sharply increases the required screening time and computational cost. Here, we introduce eBiota, a platform for ab initio design of microbial communities from a pool of 21,514 strains to generate target products. eBiota not only identifies optimal strain combinations but also simulates community behaviors, including microbial interactions and relative abundances. eBiota integrates three modules: CoreBFS, a graph-based search algorithm that rapidly screens for bacteria with complete metabolic pathways related to the target product; ProdFBA, an extended flux balance analysis that identifies microbial consortia with maximal production efficiency; and DeepCooc, a deep learning model trained on 23,323 microbiome samples across various environments to infer co-occurrence patterns. We validated eBiota’s capabilities in microbial community design and production efficiency calculation using public microbiome datasets, ranging from single strains to six-member consortia. Further in vitro experiments involving 94 strains confirmed eBiota’s ability to identify species that inhibit pathogen growth and to accurately model the relative abundances within complex microbial communities. As an initial digital twin, eBiota provides a powerful platform for the rational design of functional microbial communities, offering new opportunities for metabolic engineering and synthetic biology.

A systematic proteomic profile of oocytes from early-stage follicles, particularly primordial follicles, is critical to protect female reproductive capacity in the context of chemotherapy, yet progress has been hindered by the rarity of oocyte samples and technical challenges associated with oocyte isolation. In this study, we generated in vivo oocyte protein labeling APEX fluorescent mice. With these mice, we reconstructed the ovary in 3D, enabling precise quantification of follicles and identified 2772 proteins and 2878 gene transcripts in oocytes predominantly from primordial follicles. Proteomic shifts of short-time cisplatin treatment revealed that many altered proteins were involved in DNA damage repair and histone modification. Notably, simultaneous application of cisplatin and EZH2’s inhibitor, GSK126, relieved cisplatin-induced oocyte developmental defects. Our study provides a systematic proteomic characterization of oocytes predominantly from primordial follicles in female mice, and reveals dynamic proteome shifts in response to chemotherapeutic agents, laying the foundation for targeted fertility-preserving strategies. Using in situ APEX labeling, this study maps proteins and RNA in mouse oocytes from early ovarian follicles and shows how cisplatin alters these cells, identifying EZH2 inhibition as a potential strategy to lessen cisplatin-induced oocyte damage.

BACKGROUND:Centrosome amplification caused by Polo-like kinase 4 (PLK4) overexpression promotes tumour initiation, yet sustained PLK4 accumulation is detrimental to cancer cell viability. While proteasomal degradation limits PLK4 levels, whether alternative clearance mechanisms exist remains unknown. METHODS:We established inducible PLK4-overexpressing breast cancer cells and employed live-cell imaging, vesicular marker profiling, immunoprecipitation-mass spectrometry, and in vivo patient-derived xenograft models to investigate how cancer cells eliminate excess PLK4. FINDINGS:In this study, we identified a previously unrecognised mechanism in which excess PLK4 is rapidly expelled via migrasomes. The tetraspanin protein TSPAN6 directly binds PLK4 with high affinity and mediates its incorporation into migrasomes for extracellular release. In breast cancer samples, PLK4 expression decreased while TSPAN6 increased during progression. TSPAN6 knockdown blocked PLK4 expulsion, leading to multipolar spindle formation, apoptosis, and suppression of tumour growth and metastasis in vivo. INTERPRETATION:Migrasome-mediated clearance represents a non-proteasomal pathway maintaining centrosome homoeostasis in cancer cells. Targeting TSPAN6 to prevent PLK4 elimination selectively triggers mitotic catastrophe in PLK4-high tumours, highlighting a previously unrecognised therapeutic vulnerability. FUNDING:This work was supported by the National Natural Science Foundation of China (NSFC) (T2225006, T2488301, and 82272948 to ML), Beijing Municipal Natural Science Foundation (Key program Z220011 to ML, 5254051 to TW), NSFC (82371640 to BX, 82403693 to PW), and the "Clinic + X" program of Peking University (to PW).

Postnatal mammary gland development involves the formation of a highly branched epithelial ductal tree, primarily through the elongation and branching morphogenesis of mammary epithelial ducts. Multiple factors are involved in this process, in which both estrogen and progesterone receptors (ER/PR) play a crucial role. In this study, we identified a role of DCAF8 in promoting mammary ductal elongation and branching through its impacts on ER/PR signaling. Homozygous Dcaf8 knockout mice exhibited significant delay in mammary ductal elongation during puberty, which was characterized by a reduction in mammary ductal elongation area and distance, and terminal end buds (TEBs); abnormal branching morphogenesis of mammary ducts was also observed in adult Dcaf8 null mice, which was characterized by a reduction of lateral branches and an increase of ductal bifurcation. To further elucidate the mechanism underlying DCAF8’s role in mouse mammary development, we performed transcriptomic sequencing and biochemical experiments. The results revealed that downstream key effectors of the PR signaling pathway were significantly downregulated, while the expression of ERβ, a potential inhibitor of ERα/PR signaling, was significantly elevated in the mammary gland of Dcaf8 null mice. Collectively, this study suggests that DCAF8 may play an important role in mammary development by promoting ductal elongation and branching morphogenesis, through ERβ-mediated inhibition of ERα/PR signaling pathway.

BACKGROUND:The high mortality rate associated with epithelial ovarian cancer (EOC) is primarily due to recurrence and chemoresistance, underscoring the urgent need for innovative therapeutic approaches that leverage newly identified vulnerabilities in cancer cells. While conventional chemotherapies induce apoptosis by targeting DNA or mitotic machinery, ferroptosis represents a new distinct form of programmed cell death characterised by the accumulation of lipid peroxides. METHODS:The sensitivity of different EOC cell lines to ferroptosis inducers was evaluated using cell viability assays and lipid peroxidation measurements. Live-cell imaging with the pH-sensitive CD63-pHuji reporter was performed to track the extracellular export of acyl-CoA synthetase long-chain family member 4 (ACSL4) via exosomes. The upstream regulator of ACSL4 were identified through immunoprecipitation-mass spectrometry (IP-MS) and validated using protein binding assays. Finally, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models were utilised to evaluate the therapeutic potential overcoming ferroptosis resistance. FINDINGS:In this study, we found that interferon (IFN)-γ combined with arachidonic acid (AA), which are endogenous ferroptosis inducers, could initiate ferroptosis in most EOC cells. However, some EOC cells displayed significant resistance. Contrary to the typical increase in ACSL4 protein observed in ferroptosis-sensitive cells, resistant EOC cells exhibited surprisingly low levels of this pro-ferroptotic lipid metabolic protein. Intriguingly, this reduction is attributed to the exosomal expulsion of ACSL4 protein, revealing a distinct cellular mechanism to evade ferroptosis. We further identified VIPAS39 as a pivotal regulator in sorting ACSL4 into late endosomes, thereby facilitating their subsequent release as exosomes. Notably, targeting VIPAS39 not only overcomes the resistance to ferroptotic cell death but also markedly suppresses tumour growth. INTERPRETATION:Our findings uncover the crucial role of VIPAS39 in ferroptosis evasion by facilitating the exporting of ACSL4 protein via exosomes, highlighting VIPAS39 as a promising target for ferroptosis-based anti-cancer therapy. FUNDING:Funded by Beijing Municipal Natural Science Foundation (Key program Z220011), National Natural Science Foundation of China (NSFC) (T2225006, T2488301, 82272948), Peking University Medicine Youth Science and Technology Innovation 'Sail Plan' Project Type B Medical Interdisciplinary Seed Fund (71006Y3171), GuangDong Basic and Applied Basic Research Foundation (2021A1515110820), and the special fund of the National Clinical Key Speciality Construction Program, P. R. China (2023).

Emerging human infectious viruses originating from animals continue to pose a persistent threat to global public health. Understanding the host range of animal viruses is crucial for identifying potential spillover pathways and mitigating the risk of future pandemics. Here, we present VirHRanger, a prediction method that integrates foundation models trained on viral genome and protein sequences, alongside genomic and protein compositional traits, viral phylogeny, and protein-protein interactions. To systematically predict the animal host range, VirHRanger incorporates host taxonomy-aware neural networks trained on a comprehensive collection of animal-virus associations spanning mammals, birds, and arthropods. Within a dataset of 4,006 virus species spanning 99 viral families, our model achieved robust performance with a micro-averaged AUROC of 0.938 across all host categories, demonstrating its effectiveness in capturing generalizable host signals from viral genetic data. On a dataset of 315 novel viruses, which are associated with key reservoir animal hosts and insect vectors, VirHRanger notably outperformed the homology-based method, exhibiting a strong generalizability to novel viruses. Furthermore, VirHRanger identified host range variations among closely related viruses within the Coronaviridae family and successfully predicted the ability of SARS-CoV-2 to infect humans and other animal hosts. These findings highlight the potential of VirHRanger to transform sequencing data into timely insights for disease control during the early stages of zoonotic outbreaks. ### Competing Interest Statement The authors have declared no competing interest.

Ovarian cancer (OC) is diagnosed at advanced stages, resulting in limited treatment options for patients. While early detection of OC has been investigated, the invasiveness of approaches, high sample requirements, or false-positive rates undermined its benefits. Here, we present a “one-step” high-throughput microfluidic platform for epithelial ovarian cancer (EOC) detection that integrates small extracellular vesicle (sEV) capture, in situ lysis, and protein biomarker detection. We identified 1,818 differentially expressed proteins (DEPs) through proteomic analysis of sEVs from patients’ serum, combined with cell lines. Through multi-step screening of DEPs, we identified EOC biomarkers to customize the microfluidic platform. We used the microfluidic platform to test the expression of EOC biomarkers with 2 µL of serum from 209 participants in a prospective cohort. Based on the test results, an EOC detection model (P9) was constructed, which achieved a sensitivity of 92.3

Gene redundancy, increasing gene dosage and functional diversity, remains understudied regarding its roles in evolution and clinical infections. Exploring 22,310 prokaryotic genomes with our custom pipeline, we found that redundant genes, though less frequent than in eukaryotes, are widespread and mainly linked to niche specialization. Evolutionary analyses delineated a propensity for gene redundancy expansion with increasing phylogenetic distance, with pathogens accumulating more redundancy than non-pathogens. Redundant genes are always co-duplicated with translation initiation signals and potentially preserve functionality. Time series examining 69 Acinetobacter baumannii isolates from multi-sites in severely infected patients, we identified redundant alcohol dehydrogenase genes and translation initiation signals, introduced by gene islands, as advantageous for invasive urinary tract infection throughout its within-patient development and cross-patient transmission. Mouse peritoneal infection models and plasmid transformation experiments confirmed that the redundancy of frmA, an alcohol dehydrogenase gene, is linked to both enhanced virulence and increased biofilm mass in Acinetobacter baumannii. Additional analysis of 898 Enterobacter cloacae complex genomes revealed that redundant metal ion resistance genes carried by mobile genetic elements may provide selective advantages. This study unveiled a moderate gene redundancy within prokaryotes, providing genetic insights into the adaptive evolution and clinical infection of pathogens.

The targeted delivery of therapeutics to internal organs to, for example, promote healing or apoptosis holds promise in the treatment of numerous diseases1-4. Currently, the prevailing delivery modality relies on the circulation; however, this modality has substantial efficiency, safety and/or controllability limitations5-9. Here we report a battery-free, chipless, soft nanofluidic intracellular delivery (NanoFLUID) patch that provides enhanced and customized delivery of payloads in targeted internal organs. The chipless architecture and the flexible nature of thin functional layers facilitate integration with internal organs. The nanopore-microchannel-microelectrode structure enables safe, efficient and precise electroperforation of the cell membrane, which in turn accelerates intracellular payload transport by approximately 105 times compared with conventional diffusion methods while operating under relatively low-amplitude pulses (20 V). Through evaluations of the NanoFLUID patch in multiple in vivo scenarios, including treatment of breast tumours and acute injury in the liver and modelling tumour development, we validated its efficiency, safety and controllability for organ-targeted delivery. NanoFLUID-mediated in vivo transfection of a gene library also enabled efficient screening of essential drivers of breast cancer metastasis in the lung and liver. Through this approach, DUS2 was identified as a lung-specific metastasis driver. Thus, NanoFLUID represents an innovative bioelectronic platform for the targeted delivery of payloads to internal organs to treat various diseases and to uncover new insights in biology.

The Kirsten rat sarcoma viral oncogene homolog (KRAS) protein plays a key pathogenic role in oncogenesis, cancer progression, and metastasis. Numerous studies have explored the role of metabolic alterations in KRAS-driven cancers, providing a scientific rationale for targeting metabolism in cancer treatment. The development of KRAS-specific inhibitors has also garnered considerable attention, partly due to the challenge of acquired treatment resistance. Here, we review the metabolic reprogramming of glucose, glutamine, and lipids regulated by oncogenic KRAS, with an emphasis on recent insights into the relationship between changes in metabolic mechanisms driven by KRAS mutant and related advances in targeted therapy. We also focus on advances in KRAS inhibitor discovery and related treatment strategies in colorectal, pancreatic, and non-small cell lung cancer, including current clinical trials. Therefore, this review provides an overview of the current understanding of metabolic mechanisms associated with KRAS mutation and related therapeutic strategies, aiming to facilitate the understanding of current challenges in KRAS-driven cancer and to support the investigation of therapeutic strategies.

Ovarian cancer (OC), particularly high-grade serous ovarian carcinoma (HGSOC), is the leading cause of mortality from gynecological malignancies worldwide. Despite the initial effectiveness of treatment, acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) represents a major challenge for the clinical management of HGSOC, highlighting the necessity for the development of novel therapeutic strategies. This study investigated the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a pivotal regulator of glycolysis, in PARPi resistance and explored its potential as a therapeutic target to overcome PARPi resistance. We conducted in vitro and in vivo experiments to assess the role of PFKFB3 in OC and its impact on PARPi resistance. We analyzed PFKFB3 expression and activity in primary OC tissues and cell lines using western blotting and immunohistochemistry. CRISPR-Cas9 and pharmacological inhibitors were employed to inhibit PFKFB3, and the effects on PARPi resistance, homologous recombination (HR) repair efficiency, and DNA damage were evaluated. RNA sequencing and proximity labeling were employed to identify the molecular mechanisms underlying PFKFB3-mediated resistance. The in vivo efficacy of PARPi and PFK158 combination therapy was evaluated in OC xenograft models. PFKFB3 activity was significantly elevated in OC tissues and associated with PARPi resistance. Inhibition of PFKFB3, both genetically and pharmacologically, sensitized OC cells to PARPis, impaired HR repair and increased DNA damage. Proximity labeling revealed replication protein A3 (RPA3) as a novel PFKFB3-binding protein involved in HR repair. In vivo, the combination of PFK158 and olaparib significantly inhibited tumor growth, increased DNA damage, and induced apoptosis in OC xenografts without exacerbating adverse effects. Our findings demonstrate that PFKFB3 is crucial for PARPi resistance in OC. Inhibiting PFKFB3 sensitizes HR-proficient OC cells to PARPis by impairing HR repair, leading to increased DNA damage and apoptosis. PFKFB3 represents a promising therapeutic target for overcoming PARPi resistance and improving outcomes in OC patients.

Proteomics is transforming medical sciences, but bridging isolated samples with intact in vivo microenvironments remains a major hurdle. We present an in vivo proteomic labeling (IVPL) platform built on a new substrate, Btn-Ph-3F, and engineered ascorbate peroxidase (APEX2)-EGFPf/f mice. Btn-Ph-3F shows high stability in organs possessing complex microenvironments, while APEX2-EGFPf/f mice readily cross with commercial Cre lines, enabling specific proteomic labeling for customized cell groups in distant organs. IVPL robustly profiles in situ proteomes of intestinal epithelium, mammary gland, and tumor-infiltrating Treg cells, and, critically, labels trace exogenous proteomes from patient-derived exosomes in live mice. We identify lactate dehydrogenase A-like 6A (LDHAL6A) as a persisting exosomal effector that promotes malignant programs in recipient cells. Inhibition of LDHAL6A combined with paclitaxel treatment markedly suppresses triple-negative breast cancer growth and metastasis. Collectively, our work not only establishes an advanced model for IVPL but also profiles ultimately exosomal actors in recipient organs for targeted therapy.

Subcellular RNA localization is a conserved mechanism in eukaryotic cells and plays critical roles in diverse physiological processes including cell proliferation, differentiation, and embryo development. Nevertheless, the characterization of centrosome-localized mRNAs remains underexplored due to technical difficulties. In this study, we utilize APEX2-mediated proximity labeling to map the centrosome-proximal transcriptome, identifying DLGAP5 mRNA as a novel centrosome-localized transcript during mitosis. Using a combination of drug perturbation, truncation, deletion, and mutagenesis, we demonstrate that microtubule binding of nascent MBD1 polypeptides is required for centrosomal transport of DLGAP5 mRNA. Our data also reveal that mRNA targeting efficiency is tightly linked to the coding sequence (CDS) length. Thus, our study provides a transcriptomic resource for future investigation of centrosome-localized RNAs and sheds light on mechanisms underlying mRNA centrosomal localization.

BackgroundPoly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic lethality. However, recent clinical trials reported patients’ survival benefits from PARP inhibitor treatments, irrelevant to homologous recombination deficiency. Therefore, revealing the therapeutic mechanism of PARP inhibitors beyond DNA damage repair is urgently needed, which can facilitate precision medicine.MethodsA CRISPR-based knock-in technology was used to establish stable BRCA1 mutant cancer cells. The effects of PARP inhibitors on BRCA1 mutant cancer cells were evaluated by biochemical and cell biological experiments. Finally, we validated its in vivo effects in xenograft and patient-derived xenograft (PDX) tumour mice.FindingsIn this study, we uncovered that the majority of clinical BRCA1 mutations in breast cancers were in and near the middle of the gene, rather than in essential regions for DNA damage repair. Representative mutations such as R1085I and E1222Q caused transient extra spindle poles during mitosis in cancer cells. PAR, which is synthesized by PARP2 but not PARP1 at mitotic centrosomes, clustered these transient extra poles, independent of DNA damage response. Common PARP inhibitors could effectively suppress PARP2-synthesized PAR and induce cell senescence by abrogating the correction of mitotic extra-pole error.InterpretationOur findings uncover an alternative mechanism by which PARP inhibitors efficiently suppress tumours, thereby pointing to a potential new therapeutic strategy for centrosome error-related tumours.FundingFunded by National Natural Science Foundation of China (NSFC) (T2225006, 82272948, 82103106), Beijing Municipal Natural Science Foundation (Key program Z220011), and the National Clinical Key Specialty Construction Program, P. R. China (2023).

Abstract The treatment of ovarian cancer (OC) presents a significant challenge due to the emergence of resistance to poly (ADP-ribose) polymerase inhibitors (PARPi). Transforming acidic coiled coil containing protein 3 (TACC3) belongs to the TACC family, characterized by its coiled-coil domains. TACC3 plays a crucial role in cell division, particularly in the formation and stability of the mitotic spindle, which is essential for proper chromosome segregation. Mutations or changes in TACC3 have been associated with various cancers, making it an increasingly attractive target for anticancer therapy in recent years. Here, we demonstrated a significant upregulation of TACC3 in OC, correlating with adverse clinical outcomes. Notably, PARPi-resistant OC cells exhibited a marked sensitivity to TACC3 inhibition. This was evidenced by the induction of spindle defects and mitotic catastrophe in PARPi-resistant cells upon TACC3 suppression, achieved either genetically or pharmacologically. Moreover, we explored the therapeutic efficacy of combining TACC3 inhibitor with PARPi, specifically BO-264 with olaparib or niraparib. This combination exhibited a synergistic effect, markedly reducing cell viability in vitro and tumor growth in PARPi-resistant tumor xenograft models. Our study suggests a promising new method for treating ovarian cancer, especially for those resistant to current PARPi treatments, by simultaneously targeting TACC3 and PARPi, enhancing treatment efficacy and potentially improving patient outcomes. Citation Format: Yu Wu, Qilong Wang, Chaolin Deng, Mo Li, Yinan Xiao. Combining PARP inhibitor with TACC3 inhibition overcomes PARP inhibitor resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4757.

High mortality of ovarian cancer (OC) is primarily attributed to the lack of effective early detection methods. Uterine fluid, pooling molecules from neighboring ovaries, presents an organ -specific advantage over conventional blood samples. Here, we present a protocol for identifying metabolite biomarkers in uterine fluid for early OC detection. We describe steps for uterine fluid collection from patients, metabolite extraction, metabolomics experiments, and candidate metabolite biomarker screening. This standardized workflow holds the potential to achieve early OC diagnosis in clinical practice. For complete details on the use and execution of this protocol, please refer to Wang et al.1

Aberrant lysine lactylation (Kla) is associated with various diseases which are caused by excessive glycolysis metabolism. However, the regulatory molecules and downstream protein targets of Kla remain largely unclear. Here, we observed a global Kla abundance profile in colorectal cancer (CRC) that negatively correlates with prognosis. Among lactylated proteins detected in CRC, lactylation of eEF1A2K408 resulted in boosted translation elongation and enhanced protein synthesis which contributed to tumorigenesis. By screening eEF1A2 interacting proteins, we identified that KAT8, a lysine acetyltransferase that acted as a pan-Kla writer, was responsible for installing Kla on many protein substrates involving in diverse biological processes. Deletion of KAT8 inhibited CRC tumor growth, especially in a high-lactic tumor microenvironment. Therefore, the KAT8-eEF1A2 Kla axis is utilized to meet increased translational requirements for oncogenic adaptation. As a lactyltransferase, KAT8 may represent a potential therapeutic target for CRC.

Metastasis is a significant factor that affects the survival of patients with non-small cell lung cancer (NSCLC). Nevertheless, the molecular regulatory mechanism underlying the metastasis is currently not fully understood. This study aims to identify the important role of miR-124-3p in metastasis of NSCLC, thereby providing a potential therapeutic intervention. Exosome secretion was determined by Nanoparticle Tracking Analysis (NTA) and the uptake was measured by fluorescence inverted microscope. The binding mechanism between miR-124-3p and its upstream or downstream target genes was validated experimentally by Luciferase reporter. Cells migration was evaluated by transwell assays. Transcriptome sequencing on A549 was carried out to verify the potential signaling pathway underlying miR-124-3p regulation. Western blotting analysis was used to assess the level of AKT, p-AKT, PI3K, and p-PI3K protein expression in NSCLC cell lines. The role of miR-124-3p to suppress the tumor metastasis was verified in NSCLC xenograft model. Exosomes were more abundant in serum from patients with advanced lung cancer (n = 24 patients) than in these from patients with early-stage lung cancer (n = 30 patients), which suggested the potential correlation between amount of exosome secretion and the metastasis of NSCLC. Interestingly, the exosome release, uptake and the migration of NSCLC cells were notably inhibited by miR-124-3p. LINC00511 suppressed the expression of miR-124-3p to facilitate exosome transport due to its role as the competitive endogenous RNA for miR-124-3p. The miR-124-3p could directly target the 3′-UTR of Rab27a in NSCLC cells to inhibit exosome secretion and thereby prevent cell migration and invasion. Aside from the inhibition of exosome transport, miR-124-3p inhibited the activation of PI3K/AKT signaling in the intracellular environment. Finally, by measuring subcutaneous tumor weight and volume and lung metastasis, we also demonstrated that miR-124-3p inhibited tumor growth in vivo. In NSCLC, miR-124-3p significantly suppressed metastasis through extracellular exosome transport and intracellular PI3K/AKT signaling. These findings provide new insights toward a better understanding of the NSCLC metastasis and suggest a potential treatment biomarker for NSCLC.