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Chimeric antigen receptor T cells (CAR T cells) with T stem (T SCM ) cell-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human T SCM cell-like CAR T cells. We identified three KIs, UNC10225387B, UNC10225263A and UNC10112761A, that combined in vitro increased the frequency of CD45RA + CCR7 + TCF1 hi T SCM cell-like CAR T cells from both healthy donors and patients with cancer. KI-treated CAR T cells showed enhanced antitumor effects both in vitro and in vivo in mouse tumor models. The KI cocktail maintains T SCM cell-like phenotype preferentially in CAR T cells originating from naive T cells and causes transcriptomic changes without arresting T cell activation or modulating the chromatin organization. Specific kinases, ITK, ADCK3, MAP3K4 and CDK13, targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of T SCM cell-like CAR T cells. Knockdown of these kinases individually or in combination enriches for T SCM cell-like CAR T cells, but only CAR T cells generated in the presence of the KI cocktail show robust expansion and differentiation on stimulation with tumor cells. Overall, transient pharmacological inhibition of strategically targeted kinases maintains stem-like features in CAR T cells and improves their antitumor activity., Competing Interests: Competing interests: G.D. serves in the SAB of NanoCells, Estella, Arovella and Outspace Bio and is cofounder of Persistence Bio. A patent application has been submitted for the use of the identified KIs. Y.X. consults for Lumosa Therapeutics. The remaining authors declared no potential competing interests., (© 2025. The Author(s).)

Growing evidence supports the importance of extracellular vesicle (EV) as mediators of communication in pathological processes, including those underlying respiratory disease. However, establishing methods for isolating and characterizing EVs remains challenging, particularly for respiratory samples. This study set out to address this challenge by comparing different EV isolation methods and evaluating their impacts on EV yield, markers of purity, and proteomic signatures, utilizing equine/horse bronchoalveolar lavage samples. Horses can serve as effective translational animal models for respiratory studies due to similarities with human immune responses, shared environmental exposures, and naturally occurring respiratory diseases including asthma. Further, horses are long-lived large animals that allow for longitudinal sample collection, and provide large sample volume and cell yield, which are particularly useful since EV research is commonly limited by low sample yields. Here, EVs were isolated from horse bronchoalveolar lavage fluid (BALF) using four different methods (ultracentrifugation, microcentrifugation, and two sizes of size exclusion chromatography columns) and characterized by measuring particle counts, EV purity, total protein yield, and proteomic cargo, with a specific focus on vesicle surface marker expression potentially informing cell type of origin. We found that size exclusion chromatography yielded the highest particle counts, greatest EV purity markers and elevated vesicle surface marker expression. Overall proteomic profiles differed across isolation methods, with size exclusion chromatography clustering separately from centrifugation. Taken together, our results demonstrate that different isolation methods impact characteristics of EVs, notably that size exclusion chromatography, compared to centrifugation methods, resulted in higher EV purity and better characterized proteomic diversity, including information on EV cell-of-origin. This is the first study to characterize proteomic profiles of EVs following different isolation methods using equine BALF. The results of this study will pave the way for future studies using equine and human samples to characterize respiratory tract EVs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Hickman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Candida albicans is a growing health concern as the leading causal agent of systemic candidiasis, a life-threatening fungal infection with a mortality rate of ~40% despite best available therapy. Yck2, a fungal casein kinase 1 (CK1) family member, is the cellular target of inhibitors YK-I-02 (YK) and MN-I-157 (MN). Here, multiplexed inhibitor beads paired with mass spectrometry (MIB/MS) employing ATP-competitive kinase inhibitors were used to define the selectivity of these Yck2 inhibitors across the global C. albicans proteome. The MIB matrix captured 89% of the known and predicted C. albicans protein kinases present in cell lysate. In MIB/MS competition assays, YK and MN demonstrated exquisite selectivity across the C. albicans fungal kinome with target engagement of only three CK1 homologs (Yck2, Yck22, and Hrr25) and a homolog of human p38α (Hog1). Additional chemoproteomics using a custom MN-kinobead identified only one additional C. albicans protein, confirming its remarkable fungal proteome-wide selectivity. To identify new Yck2 inhibitors with selectivity over Hog1, thirteen human CK1 kinase inhibitors were profiled for fungal kinase-binding activity using MIB/MS competition assays and in-cell NanoBRET target engagement assays. A new chemotype of family-selective Yck2 inhibitors with antifungal activity was identified. Together, these findings expand the application of MIB/MS proteomic profiling for non-human kinomes and demonstrate its utility in the discovery and development of selective inhibitors of fungal kinases with potential antimicrobial activity.

The DNA damage response (DDR) mechanisms that allow cells to tolerate DNA replication stress are critically important for genome stability and cell viability. Using an unbiased genetic screen we identify a role for the RING finger E3 ubiquitin ligase RNF25 in promoting DNA replication stress tolerance. In response to DNA replication stress, RNF25-deficient cells generate aberrantly high levels of single-stranded DNA (ssDNA), accumulate in S-phase and show reduced mitotic entry. Using single-molecule DNA fiber analysis, we show that RNF25 protects reversed DNA replication forks generated by the fork remodeler HLTF from nucleolytic degradation by MRE11 and CtIP. Mechanistically, RNF25 interacts with the replication fork protection factor REV7 and recruits REV7 to nascent DNA after replication stress. The role of RNF25 in protecting replication forks is fully separable from its canonical functions in ubiquitin conjugation. This work reveals the RNF25-REV7 signaling axis as an important protective mechanism in cells experiencing replication stress., Competing Interests: Declaration of Interests The authors declare no competing interests.

Wildfires adversely impact air quality and public health worldwide. Exposures to wildfire smoke are linked to adverse health outcomes, including cardiopulmonary diseases. Critical research gaps remain surrounding the underlying biological pathways leading to wildfire-induced health effects. The regulation of intercellular communication and downstream toxicity driven by extracellular vesicles (EVs) is an important, understudied biological mechanism. This study investigated EVs following a wildfire smoke-relevant in vitro exposure. We hypothesized that woodsmoke (WS) would alter the proteomic content of EVs secreted in organotypic in vitro airway models. Exposures were carried out using a tri-culture model of alveolar epithelial cells, fibroblasts, and endothelial cells and a simplified co-culture model of alveolar epithelial cells and fibroblasts to inform responses across different cell populations. Epithelial cells were exposed to WS condensate and EVs were isolated from basolateral conditioned medium following 24 h exposure. WS exposure did not influence EV particle characteristics, and it moderately increased EV count. Exposure caused the differential loading of 25 and 35 proteins within EVs collected from the tri- and co-culture model, respectively. EV proteins involved in extracellular matrix degradation and wound healing were consistently modulated across both models. However, distinct proteins involved in the wound healing pathway were altered between models, suggesting unique but concerted efforts across cell types to communicate in response to injury. These findings demonstrate that a wildfire-relevant exposure alters the EV proteome and suggest an impact on EV-mediated intercellular communication. Overall, results demonstrate the viability of organotypic approaches in evaluating EVs to investigate exposure-induced biomarkers and underlying mechanisms. Findings also highlight the impact of differences in the biological complexity of in vitro models used to evaluate the effects of inhaled toxicants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)