Intracellular nucleotide imbalance between ribonucleotides and deoxyribonucleotides can trigger cytosolic mitochondrial DNA (mtDNA) release and subsequent activation of the cGAS-STING pathway. However, fluoropyrimidine-induced nucleotide imbalance is constrained by their metabolic instability. Here, we leverage ribonucleotides to amplify floxuridine (FUDR)-induced mtDNA release and cGAS-STING activation for enhanced cancer chemoimmunotherapy. We identified that cytidine monophosphate (CMP) significantly enhances FUDR-mediated activation of the mtDNA-driven cGAS-STING pathway, inducing over 300-fold upregulation of the interferon gene Ifnb1 and initiating STING-dependent antitumor immunity. Genome-wide CRISPR-Cas9 screening identified five pyrimidine-metabolizing enzymes (CMPK1, CMPK2, TYMP, UPP1, and UPP2) that are responsible for the metabolic instability of floxuridine monophosphate (FdUMP). CMP and its intracellular metabolites can metabolically block these enzymes, thereby increasing the peak intracellular concentration of FdUMP by over 500-fold and extending its half-life by more than 20-fold, which in turn promotes sustained intracellular nucleotide imbalance. To translate this mechanism into therapy, we co-encapsulated FdUMP and CMP into hybrid lipid nanoparticles (FC-NPs). FC-NPs robustly activated STING-dependent antitumor immunity, achieving remarkable therapeutic efficacy in multiple murine colorectal cancer (CRC) models. Our study not only addresses the long-standing issue of fluoropyrimidine metabolic instability but also reveals a novel ribonucleotide-mediated immunomodulatory mechanism, providing a promising strategy to potentiate chemoimmunotherapy in CRC.

The standard of care for patients with unresectable or advanced cholangiocarcinoma (CCA) is systemic chemotherapy combined with immunotherapy, which achieves a median overall survival (OS) of 13 months and a 3-year OS of 15%. This provides the benchmark against which additional locoregional therapies are evaluated. This narrative literature review examined locoregional therapies, their clinical outcomes, and recommendations for their role in the management of patients with CCA. The review focused on intrahepatic (iCCA) and perihilar CCA (pCCA) with locoregional disease in patients who were ineligible for surgical resection. A literature search was conducted in PubMed (MEDLINE) to identify clinical studies evaluating locoregional therapies in CCA. Ongoing and completed trials were identified through ClinicalTrials.gov. For iCCA, clinical trials have evaluated percutaneous image-guided local ablative therapies, including radiofrequency ablation or microwave ablation (3-year OS 42%), cryoablation, interstitial electroporation, and brachytherapy. Other trials have analysed external beam radiotherapy (EBRT), often using high-precision techniques (3-year OS 38%), as well as intra-arterial therapies, including transarterial chemoembolisation (3-year OS 17%), selective internal radioembolisation (3-year OS 40%), and floxuridine delivery through a hepatic arterial infusion pump (3-year OS 26-50%). For pCCA, EBRT has been evaluated in three treatment settings: definitive therapy (1-year OS 100%), adjuvant postoperative therapy (3-year OS 58%), and neoadjuvant therapy before liver transplantation (3-year OS 62%). Recent and ongoing clinical trials demonstrate a clear trend toward combining locoregional and standard-of-care systemic therapies. Few randomised controlled trials have been conducted. Until further evidence from randomised controlled trials becomes available, treatment strategies for patients with unresectable or advanced CCA should be individualised, incorporating flexible, staged combinations of locoregional and systemic therapies within a personalised therapeutic framework. These decisions should be made within a multidisciplinary tumour board.

The Caenorhabditis Intervention Testing Program recently characterized the longevity-promoting effects of all-trans-retinoic acid (atRA) in C. elegans . While we observed that atRA extended lifespan across multiple genetic backgrounds, environmental factors may influence its efficacy. One such factor is 5-Fluoro-2'-deoxyuridine (FUdR), used to prevent progeny contamination in lifespan assays. Here, we show that FUdR enhances atRA's lifespan effects, but that this potentiation is not simply due to progeny elimination, as sterilization through auxin-mediated disruption of spermatogenesis does not potentiate atRA in lifespan extension. We conclude that atRA longevity is dependent on additional effects of FUdR that remain uncharacterized.

Fasting-based dietary interventions are conserved regulators of aging that extend lifespan across species, including Caenorhabditis elegans. However, fasting studies in C. elegans are sensitive to experimental variables that can independently influence lifespan and health, including FUdR, antibiotic treatment, germline-less mutants, and the use of UV- or heat-killed bacteria. FUdR can alter lifespan, age-associated pathology, and stress responses, while antibiotics used to prevent bacterial growth during fasting may directly affect worm physiology. To minimize these confounding factors, we developed a simple adult-onset intermittent fasting paradigm that does not require FUdR, antibiotics, or bacterial killing. Wild-type worms were subjected to daily fasting periods of 5 h, 6 h, or 18 h until day 10 of adulthood and compared with continuously fed controls. Daily intermittent fasting robustly extended lifespan by 24-57%, demonstrating that repeated fasting windows during adulthood are sufficient to promote longevity under minimally confounded conditions. These findings establish a straightforward and experimentally tractable intermittent fasting paradigm for C. elegans and underscore the importance of limiting pharmacological and microbial conditions in dietary-intervention experiments.

Protozoan parasites remain a major threat to global health, affecting both humans and livestock. Theileria annulata, an apicomplexan parasite of cattle, transforms host leukocytes into a cancer-like state, while Leishmania donovani causes visceral leishmaniasis in humans. Given their reliance on host signaling and metabolic pathways, we hypothesized that FDA-approved anticancer drugs could be repurposed as antiparasitic agents. A library of 436 compounds was screened against T. annulata, yielding 130 hits at 10 µM, of which 63 exhibited minimal cytotoxicity toward healthy PBMCs. Secondary screening at 1 µM identified 28 potent inhibitors with > 50% activity. These compounds span diverse scaffolds, including taxanes, nucleoside analogues, antifolates, proteasome inhibitors, and kinase modulators, and target critical pathways such as apoptosis, autophagy, DNA damage response, cytoskeletal dynamics, NF-κB signalling, and tyrosine kinase activity. Notably, floxuridine, masitinib, paclitaxel, cabazitaxel, vinorelbine, maytansine, and bortezomib also suppressed L. donovani, highlighting cross-species efficacy. Target mapping revealed that the majority of active molecules disrupt hallmarks of cancer and parasite-transformed cells, including metabolic reprogramming, genome instability, and invasive phenotypes. Collectively, these findings underscore the value of oncology-derived drug libraries for antiparasitic discovery and identify promising candidates for further evaluation against T. annulata and L. donovani.

Efforts to improve the effectiveness of existing interventions for antimicrobial-resistant infections include identifying new ways to overcome resistance to licensed antibiotics using adjuvants or deploying antibiotics in novel combinations. Although antibiotics targeting the bacterial cell wall (e.g., β-lactams) and folate metabolism (e.g., trimethoprim-sulfamethoxazole [TMP-SMX]) remain cornerstones of modern healthcare, resistance to both classes poses an ongoing therapeutic challenge. We recently demonstrated that purine nucleosides can act as potent antibiotic adjuvants, restoring β-lactam susceptibility in methicillin-resistant Staphylococcus aureus (MRSA). Here, we show that guanosine significantly reduces intracellular thymidine levels in MRSA and potentiates the activity of antifolate antibiotics (TMP-SMX), as well as the pyrimidine antimetabolites 5-fluorouracil and 5-fluorouridine. Incorporation of oxacillin into guanosine-antifolate or guanosine-pyrimidine analog combinations further enhanced killing of both planktonic and biofilm-associated MRSA. Thymidine depletion was accompanied by elevated intracellular reactive oxygen species and dissipation of membrane potential, providing mechanistic insight into the bactericidal effects of these combinations. Together, these findings demonstrate that guanosine expands MRSA susceptibility beyond β-lactams to include additional clinically relevant antimicrobial drug classes commonly used to treat bacterial infections.

Cupriavidus necator H16 is a promising chassis for one-carbon (C1) biomanufacturing due to its ability to assimilate CO2 with H2-derived reducing power, yet genome engineering in this bacterium remains constrained by inefficient DNA delivery and low homologous recombination efficiency. Here, we establish a modular two-plasmid CRISPR/Cas9 recombineering system that decouples Cas9 expression from sgRNA turnover, incorporates a self-splicing intron riboswitch to tightly control Cas9 counterselection, and introduces a pair of bacteriophage recombinases, Che9c60 and Che9c61, to markedly enhance homologous recombination efficiency. With Che9c60 and Che9c61, the gene knockout efficiency increased from undetectable levels to 58.3% and reached 100% at certain loci. Importantly, introducing these two recombinases enabled scarless chromosomal knock-in by boosting insertion efficiency from undetectable levels to nearly 40%, and supported targeted integration of DNA payloads up to 4.0 kb. To facilitate rapid multiround engineering, we integrated a tdk-based counterselection marker into the sgRNA plasmid, reducing plasmid-retaining cells to 1.1% within 24 h under 5-fluoro-2'-deoxyuridine (FUDR) selection. Overall, this streamlined toolkit enables efficient, iterative, and scarless genome editing in C. necator, accelerating C. necator strain development for C1 metabolic engineering and industrial applications.

Off-patent, generic drugs are expected to be widely accessible owing competitive pricing and minimal R&D costs. Sudden, excessive, unexplainable, and sustained price increases detrimentally impact the health and welfare of patients and their families. Using Medicare Part B quarterly payment data over 2020-2025, we observed a sudden, steep and sustained increase of over 5000% in the price of floxuridine injection, with the most noticeable spike between October 2022 and January 2023. Comparable increases in magnitude were also corroborated in state Medicaid programs. Plausible explanations, including raw material shortage, regulatory changes, formulation changes, and change in patent status, did not support the steep price hike. Market exit of manufacturer(s) to the point of exclusivity appears to be the only reasonable explanation for the price hike. While legally permissible, such opportunistic pricing of floxuridine undermines the principle of generic drug accessibility. This case reflects a broader concern that generic oncology drugs may be subject to morally questionable pricing. As floxuridine becomes more widely available through ongoing clinical trials, concerns about its affordability and financial toxicity will be difficult to ignore. Floxuridine serves as a cautionary tale of how niche-market generics can evade public scrutiny while imposing unnecessary costs on our healthcare system.

Ferroptosis has emerged as a viable approach to enhance therapeutic efficacy in triple-negative breast cancer (TNBC), making monitoring essential for prognostic evaluation. However, conventional MR imaging cannot characterize ferroptosis-related biomarkers. Herein, we propose a Relaxation Suppressed Exchange Tuning (ReSET) MRI strategy for ferroptosis modulation and monitoring, which relies on T1 relaxation and chemical exchange saturation transfer (CEST). ReSET was achieved by a tumor-microenvironment-responsive nanoprobe (SiO2@FUDR@MnO2, SiFM), consisting of a mesoporous silica core loaded with chemotherapeutic floxuridine and the MnO2 shell with nanozyme activities. This structure promotes robust ROS generation, which induces lipid peroxidation and ultimately facilitates chemodynamic therapy (CDT)-sensitized ferroptosis. Notably, ReSET takes advantage of the tumor-responsive release of Mn2+ from SiFM, which shortens T1 relaxation and suppresses the saturation transfer efficiency between water and exchangeable endogenous protons reducing magnetization transfer ratio asymmetry (MTRasym). The inverse correlation between the T1 intensity and MTRasym provides a sensitive and specific indicator for visualizing glutathione peroxidase 4 (GPX4) expression, thereby enabling cross-modal monitoring of ferroptosis. Meanwhile, we revealed that the ReSET strategy enables sensitive detection of small lesions in liver micrometastasis models. Overall, we establish a ReSET MRI-guided strategy using a manganese-based nanoplatform, which represents a promising ferroptosis-related theranostic approach for TNBC.

Many commonly used therapeutic drugs cause biliary toxicity, but it is unclear if they are directly responsible for the increasing incidence of cholangiocarcinoma (CCA). We tested experimentally and analyzed through a cohort approach whether drugs, such as the commonly used antibiotic Augmentin, which is a poster-child of biliary toxicity, are causally linked to CCA development. Using sophisticated analytical tools in cholangiocytes, including single extracellular vesicle (EV) analysis, we found no evidence that Augmentin increases the cholangiocyte malignancy marker YAP1 or phospho-YAP1. Furthermore, we analyzed the CCA incidence in our healthcare system and determined it to be 0.0932% (Augmentin group) and 0.0799% (amoxicillin control group). Although the Augmentin group showed a numerically higher CCA incidence, the association did not reach statistical significance (RR = 1.1669, 95% CI 0.6200-2.1961; Fisher's exact test, P = 0.7493). Similarly, we found no evidence for cholangiocarcinoma development with other commonly used drugs, including chlorpromazine, floxuridine, 5-fluorouracil, flucloxacillin and terbinafine. We conclude that there is no direct causal relationship between clinical Augmentin doses and CCA development.