Extracellular vesicles (EVs) play an important role in cancer progression and metastasis. The increasing clinical proteomics data provides an opportunity to uncover new biomarkers and therapeutic targets in cancer specific EVs. Here, we present an integrated data analysis approach that leverages a comprehensive protein catalog from EVs, blood plasma, and the cancer cell surface to identify EV-associated proteins and evaluate their potential as therapeutic targets in cancer cells. Quantitative proteomics data from 12 cancer types (n = 2,272) identified 3,250 proteins that are commonly found in EV studies and EV quantity data. Receptor kinases (RKs) are crucial in cancer signalling pathways and have been widely studied targets. Our analysis focused on identifying RKs with significant high expression in cancer types that were also detected in EVs. Notably, PTK7 emerged as a significant target with high expression or survival association in seven independent cancer cohorts. We then explored the druggability of the RKs. Our analysis of drug repurposing network and RNAi screening suggested PTK7 as a potential therapeutic target with no FDA approved drugs in cancer. Overall, this integrative analysis proposes a framework to prioritize EV-associated candidates for downstream therapeutic investigation in cancer.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe, often fatal, disease characterized by hyperinflammation and features of a cytokine storm. Hyperproduction of both IL-10 and IL-6 and low TGF-β production can generate a cytokine storm, with IL-10 playing a particularly important role. To investigate the role of IL‐10 in patients with SFTS, we analyzed the phenotypes of macrophages, cytokines, and signaling pathways in patients with mild to fatal SFTS and found that the population of HLA-DR+CD86+ macrophages was increased, the population of CD163+CD206+ macrophages was decreased, the levels of IL-10 (p < 0.0001), IL-6 (p < 0.0001), TNF-α (p=0.1056), and CCL1 (p=0.1533) were increased, TGF-β (p=0.0104) was increased, and Smad3 and P-Smad3 were highly expressed in patients with fatal SFTS. We also investigated the role of IL‐10 in THP‐1-derived macrophages infected with SFTSV or SARS‐CoV‐2, treated with lipopolysaccharide (LPS), or treated with serum from patients with fatal SFTS. We found that blocking IL‐10 signaling can decrease the population of HLA-DR+CD86+ cells, increase the population of CD163+CD206+ cells, reduce IL‐6 and TNF-α production, increase TGF‐β production and induce the expression of Smad3 and P-Smad3 in SFTSV- and SARS‐CoV‐2‐infected and LPS‐induced THP-1 cells. Additionally, IL-10 receptor blockade can reduce IL-10 and IL‐6 production in THP-1 cells treated with serum from patients with fatal SFTS. Therefore, we suggest that HLA-DRhiCD86hi macrophages may contribute to pathological activity and that CD163hiCD206hi macrophages may play a critical role in the protection of effector functions against SFTSV and SARS-CoV-2 infection. IL-10 could serve as a prognostic target, and antibodies against the IL-10 receptor could represent a potential immune-based intervention against a cytokine storm in patients with fatal SFTS and severe/critical COVID-19.

Long-term culture of human pluripotent stem cells (hPSCs) can lead to spontaneous mutations, genomic abnormalities, and alterations in gene expression, thereby compromising their self-renewal and pluripotency. Thus, optimizing the long-term culture conditions of hPSCs is crucial. In this study, we introduce O-cyclic phytosphingosine-1-phosphate (cP1P, Axceso Biopharma Co. Ltd.), a novel culture additive structurally analogous to S1P, which markedly enhances hPSC self-renewal and survival. Our results demonstrate that cP1P supplementation promotes long-term proliferation of hPSCs by upregulating pluripotency markers and maintaining their ability to differentiate into cell types derived from the three germ layers. Furthermore, RNA-seq analysis reveals that cP1P alleviates long-term culture-induced upregulation of apoptosis- and chordate embryonic development-related genes, while preventing the downregulation of stem cell maintenance pathways. Collectively, these findings suggest that cP1P effectively supports the proliferation, pluripotency, and differentiation potential of hPSCs during both short- and long-term cultures.

Polyhexamethylene guanidine phosphate (PHMG-p), a cationic disinfectant previously used in humidifiers, has been linked to severe pulmonary diseases in Korea. This study aimed to elucidate the molecular mechanisms underlying PHMG-p–induced lung toxicity using an integrated multi-omics approach. BALB/c mice were intratracheally instilled with PHMG-p (0, 0.03, 0.1 mg/kg, twice weekly for 4 weeks). Histopathology revealed dose-dependent pulmonary lesions, including inflammatory infiltration, alveolar wall hyperplasia, and fibrosis. Transcriptomic profiling identified 213 and 1,506 differentially expressed genes (DEGs) in the low- and high-dose groups, respectively, with enriched pathways related to immune activation, cytokine signaling, and cellular stress responses. Proteomic analysis detected 148 and 1,168 differentially expressed proteins (DEPs), many of which overlapped with DEGs and were associated with chemokine signaling, protein refolding, and ion transport dysregulation. Metabolomic profiling of serum samples identified dose-responsive alterations in amino acid and energy metabolism, with notable increases in glutamate, leucine, serine, and related metabolites. Integrated omics analysis revealed consistent up-regulation of CDKN1A, HSP90AA1, HSPA1A, HSPA8, and HSPH1, and down-regulation of FPR1, suggesting their roles as potential biomarkers of PHMG-p–induced pulmonary injury. Pathway convergence indicated activation of inflammatory and fibrotic remodeling processes, as well as metabolic reprogramming involving glutamate and branched-chain amino acid pathways. These findings provide mechanistic insight into PHMG-p–induced lung toxicity and highlight multi-omics signatures that may serve as biomarkers for monitoring or predicting pulmonary damage caused by cationic polymer biocides.

Plants adapt to winter through two key strategies: vernalization, which enables flowering after prolonged cold, and cold acclimation, which enhances freezing tolerance. Although both require long-term cold perception, how they are integrated remains unclear. We identify the cytosolic chaperonin TCP1 Ring Complex (TRiC)/Chaperonin Containing TCP1 (CCT) complex as a critical upstream regulator of both processes in Arabidopsis. A missense mutation in CCT8 impairs vernalization and freezing tolerance, with reduced expression of VERNALIZATION INSENSITIVE 3 (VIN3) and C-REPEAT BINDING FACTORs (CBFs), central regulators of these processes. TRiC is required for the accumulation of REVEILLE (RVE) transcription factors, core components of the circadian oscillator. In particular, RVE8 activates VIN3 and CBF expression by binding their promoters. Genetic and biochemical evidence shows that TRiC promotes RVE abundance during cold exposure, thereby contributing to VIN3-associated epigenetic silencing of FLOWERING LOCUS C and CBF-dependent freezing tolerance. VIN3 expression is also circadian-regulated and preferentially induced by cold during the subjective day, revealing circadian gating. These findings uncover a TRiC-RVE-VIN3/CBF regulatory framework linking circadian signaling with seasonal cold adaptation, and show how a core protein-folding machinery modulates transcription factors to coordinate developmental processes and stress responses in winter.

Adipocyte differentiation from mesenchymal stem cells is governed by tightly regulated transcriptional and epigenetic programs. The Hippo pathway effectors YAP and TAZ impede this process, yet the underlying molecular mechanisms remain unclear. Here, we demonstrate how YAP/TAZ regulate transcription of the adipocyte lineage specification factor PPARγ and its target genes at the chromatin level. TAZ represses PPARγ-bound target enhancers as evidenced by a markedly reduced histone H3 acetylated at lysine-27 occupancy, resulting in the transcriptional repression of adipogenic genes, including Pparg2. Single-nucleus genomic analyses of mouse adipose tissue further revealed that YAP/TAZ activation drives extensive epigenetic modulation. Notably, the repressive effect of TAZ on adipogenic enhancers requires TEAD-dependent transcriptional activity, but not a direct interaction with PPARγ through the WW domain as previously reported. Last, we identified Vestigial-like 3 (Vgll3) as a transcriptional target of TAZ critical for repressing adipogenic enhancers. These findings reveal that the YAP/TAZ-VGLL3 axis regulates adipocyte fate by repressing the PPARγ program at its target enhancers.

Understanding of dynamic activations of pathophysiological processes in the infected lungs is important for effective treatment of SARS-CoV-2 infection. Time-course transcriptome analyses of infected lungs have been performed to address this issue. Since proteins actually execute the pathophysiological processes, however, the time-course transcriptome data provide limited information regarding their temporal transitions. Here, we present time-course proteomic profiling of lung tissues from K18-hACE2 transgenic mice using liquid chromatography-tandem mass spectrometry analysis at day 0, 1, 2, 5, and 7 after SARS-CoV-2 infection. Clustering analysis to identify early, intermediate, and late up-regulated proteins, analysis of pathways enriched by these proteins, and network analysis of early, intermediate, and late up-regulated pathways revealed detailed dynamic activations of molecular networks perturbed upon infection, and further proposed five markers that represented early-to-intermediate activation of nonsense-mediated decay (Smg6 and Upf1), intermediate-to-late activation of phagocytosis (Fcgr4 and Lamp2), and late activation of neutrophil extracellular trap formation (Padi4) that could be associated with severe pathological transitions. Immunohistochemistry analysis confirmed these temporal up-regulation patterns of the five markers identified from the time-course proteome data, and immunofluorescence analysis further confirmed that Padi4 was up-regulated predominantly in neutrophils at the late stage. Our temporal proteomic analysis suggests potential pathway and molecule markers that can be used to predict severe pathological transitions during the course of SARS-CoV-2 infection.

Abstract Current treatment strategies for medulloblastoma remain ineffective owing to extensive tumor heterogeneity. We generated five platforms of omics data including liquid chromatography and mass spectrometry-based proteome and performed integrated multi-omic characterization to improve the conventional molecular classification of medulloblastoma. We identified seven refined distinct subtypes. The sonic hedgehog (SHH) group was reclassified into two subgroups, SHHα and SHHβ, whereas group 4 was divided into three subgroups, G4α, G4β, and G4γ. SHH and group 4 subtypes exhibit two distinct neuronal differentiation trajectories: granular neuron and unipolar brush cell differentiation (SHHβ and G4γ, respectively), both of which associated with more favorable clinical outcomes. Furthermore, we uncovered unique proteomic and kinomic properties that conferred increased treatment vulnerabilities to targeted therapeutic interventions against each of the three medulloblastoma subtypes associated with poor clinical outcomes. We demonstrated the therapeutic potential of exploiting these vulnerabilities by utilizing a proteasome inhibitor and subtype-specific agents, including CDK1/2, PARP, CLK1, and MET inhibitors. Mechanistic insights were further elucidated through in-depth proteome analyses. Our study qualifies the use of proteomic signatures and activation of neuronal differentiation trajectories to tailor selective therapeutic opportunities for distinct subgroups of patients with medulloblastoma.

Photosynthetic protein complexes in thylakoid membranes disassemble during leaf senescence, leading to a decline in photosynthetic efficiency. However, the mechanisms by which these protein complexes in chloroplast thylakoid membranes modulate leaf senescence are poorly understood. This study investigated the roles of Arabidopsis thaliana STN7 and STN8 kinases during leaf senescence using proteomic tools, characterizing STN7 and STN8 overexpressors and loss-of-function mutants. During leaf senescence, proteins involved in photosynthesis-associated processes decreased, while those related to lipid metabolism and jasmonic acid-mediated stress responses increased. The abundance and phosphorylation of the thylakoid membrane proteome were regulated by STN7, but not by STN8. Thirty-four STN7 target proteins overlapped with senescence-associated proteins in the wild type, most of which are related to the photosynthetic protein apparatus. Three out of nine mutants defective in STN7 downstream targets (lfnr2, tsp9, and riq2) exhibited accelerated leaf senescence based on chlorophyll a/b ratio measurements and photochemical analyses. Photosynthetic complex formation was normal in all STN7 target protein loss-of-function mutants, indicating the absence of feedback regulation upstream of STN7 during photosynthetic complex assembly. Overall, the data reveal that STN7 is a major kinase component in signaling cascades that link photosynthetic complex formation and leaf senescence.

Inherited mutation in breast cancer susceptibility gene 1 (BRCA1) is strongly associated with mammary tumors that exhibit triple-negative characteristics, are insensitive to endocrine-targeted therapies, and show basal-like properties, including aggressive phenotypes [1, 2]. It has been reported that the average cumulative risk of breast cancer for BRCA1 mutation carriers by age 70 years is 57% (95% confidence interval [CI]: 47%-66%) [3]. Despite the high incidence and aggressive characteristics of BRCA1-associated breast cancer, few substantial improvements in preventing or treating this cancer have been made, largely due to the challenges of clinic-based cohort studies. During malignant transformation, cancer progression is facilitated by metabolic reprogramming–one of the hallmark characteristics of cancer. Previously, we found that inhibition of AKT is a potential strategy for the prevention and therapeutic management of Brca1-mutant mammary tumors. However, pharmacological inhibition proved less effective and less safe compared to genetic perturbation, limiting its potential for clinical application [4]. Meanwhile, mTOR, a key regulator of metabolism and a downstream target of the PI3K/AKT signaling pathway, has emerged as a promising therapeutic target for several diseases, including treatment of cancer [5]. In addition to identifying the contribution of mTOR signaling to BRCA1-deficient cells (Supplementary Figure S1), we provide genetic and pharmacological evidence using multi-orthogonal preclinical models [6-8] that mTOR is closely involved in the development and growth of Brca1-mutated mammary tumors (Figure 1A). To investigate the role of mTOR in the absence of BRCA1, we assessed the development of mammary glands in post-pubertal Brca1/Mtor-mutant mice by examining ductal and lobular development of the fourth mammary gland. Measurements of mammary gland density using the Branch software (ver. 1.1 [9]) showed that ductal length and branching were significantly diminished in the mammary glands of Brca1co/coMtorco/coMMTV-Cre mice (Figure 1B,C, Supplementary Figure S2). To determine whether mTOR contributes to BRCA1-deficient mammary tumor formation, we examined tumor formation in cohorts of Brca1co/co (n = 28), Brca1co/coMtorco/co (n = 30), Brca1co/coMMTV-Cre (n = 24), and Brca1co/coMtorco/coMMTV-Cre (n = 29) mice (Top left of Figure 1A). Brca1co/co and Brca1co/coMtorco/co mice showed no signs of mammary abnormalities, including tumors, up to 24 months of age. In contrast, Brca1co/coMMTV-Cre mutant mice developed breast cancer, reaching a high incidence (37.5%; 9/24) by 24 months of age. During the same period, Brca1co/coMtorco/coMMTV-Cre mice exhibited a lower incidence of breast cancer (6.9%; 2/29) and significantly better tumor-free survival compared to Brca1co/coMMTV-Cre mice (P = 0.008, log-rank test) (Figure 1D). Next, we examined whether mTOR inhibition using a clinically applicable pharmacological approach would produce similar effects as genetic ablation. To test pharmacological inhibition of mTOR, we administered everolimus (20 mg/kg, oral, 5 times/week) or vehicle to 4-month-old Brca1co/coMMTV-Cre mice for 11 months (lower left of Figure 1A, Supplementary Figure S3). During this period, Brca1co/coMMTV-Cre mice in both groups spontaneously developed palpable mammary tumors. At the end of the study period (15 months of age), vehicle-treated Brca1co/coMMTV-Cre mice showed a high incidence of mammary tumors (93%; 13 of 14). During the same period, everolimus-treated Brca1co/coMMTV-Cre mice exhibited a breast cancer incidence of 46% (5 of 11) and significantly longer tumor-free survival compared to their vehicle-treated counterparts (P = 0.0117, log-rank test) (Figure 1E). Moreover, while multiple tumors were found in 2 of 14 (14%) vehicle-treated mice, no cases of multiple tumors were detected in everolimus-treated mice (Figure 1F). In addition to tumor formation, whole-mount analysis of non-tumor–bearing mammary glands revealed that everolimus treatment reduced total ductal length and branch number by approximately 30% compared to vehicle treatment (Supplementary Figure S3). Notably, everolimus treatment significantly reduced the formation of abnormal hyperplastic foci (0.6 vs. 5.5 foci/mammary gland; P < 0.01) in non-tumor–bearing mammary glands of Brca1-mutant mice (Figure 1G,H). Taken together, these results suggest that genetic ablation and pharmacological inhibition of mTOR signaling prevents the proliferation of mammary epithelial cells and reduce tumor formation in Brca1-mutant mice. Contribution of mTOR in the initiation and progression of BRCA1-associated mammary tumors. (A) Overview of the experimental designs to investigate the attenuation of tumor initiation and progression by mTOR inhibition in Brca1-mutant mice. (B) Representative whole-mount staining of mammary glands from 2-month-old mice with the indicated genotypes. Arrows indicate the presence of sprouting ducts in the mammary glands. Scale bar, 5 mm. (C) Total length (light gray) and branch numbers (dark gray) of ducts between the lymph node and the end tip in the 4th mammary glands of 2-month-old Brca1co/co (n = 9), Brca1co/coMtorco/co (n = 16), Brca1co/coMMTV-Cre (n = 18), and Brca1co/coMtorco/coMMTV-Cre (n = 25) mice, estimated using Branch software (**P < 0.01, one-way ANOVA with Tukey's post hoc correction). (D) Kaplan-Meier curves of tumor-free survival showing normal survival (100%) of Brca1co/co (n = 28) and Brca1co/coMtorco/co (n = 30) mice and significantly diminished tumor-free survival in Brca1co/coMMTV-Cre mice (n = 24) compared with Brca1co/coMtorco/coMMTV-Cre mice (n = 29) (*P = 0.008, log-rank test). (E-F) Kaplan-Meier curves (E) and Summary data (F) showing tumor-free survival of Brca1co/coMMTV-Cre mice treated with vehicle (n = 14) or everolimus (EVE, n = 11, 20 mg/kg, oral, 5 times/week). (G) Representative whole-mount staining of non-tumor-baring mammary glands from 15-month-old Brca1co/coMMTV-Cre mice treated with vehicle or everolimus for 11 months. Small arrowheads indicate abnormal foci. The panels on the right are magnifications of the boxed areas in adjacent panels. Scale bar, 5 mm. (H) Number of foci per mammary gland in vehicle- (n = 6) and everolimus-treated (n = 11) non-tumor-baring mammary glands (**P < 0.01). (I) Upon spontaneous tumor appearance, tumor-bearing mice were randomized into vehicle (n = 11) or everolimus (n = 15, 20 mg/kg, oral, 5 times/week) treatment. Tumor growth progression was monitored weekly by MRI. Representative MRI scans of tumor-bearing mice at baseline and following the indicated treatments. (J) Graphs showing RTVs (left panel) between post-treatment and baseline (start of treatment) and analysis of weekly progression (right panel). Tumor growth was assessed by the RTV = tumor volume at a given time (cm3)/tumor volume at the initiation of treatment (cm3). Weekly progression in the everolimus-treated group was significantly lower than that in the vehicle-treated group (P = 0.0002, chi-square test). (K) Summary data showing tumor progression and mouse survival following vehicle or everolimus treatment. (L) Responsiveness of spontaneously developed mammary tumors from Brca1co/coMMTV-Cre mice to everolimus, segregated based on mouse survival: non-responder (NR), survival ≤ 9 weeks; responder (R), survival > 9 weeks. (M) Representative MRI scans of non-responder and responder tumor-bearing mice at baseline and the indicated times. (N) Summary data showing everolimus-responsiveness of tumor progression (RTV) and mouse survival. (O) Graph showing calculated RTVs for engrafted tumors treated with vehicle (black lines) or everolimus (red lines). Spontaneously developed mammary tumors (n = 22) were collected from Brca1co/coMMTV-Cre mice and transplanted into nude mice. Growth of the corresponding tumors in sham-treated mice versus mice treated with everolimus (5 mg/kg, oral, 5 times/week) was tested. All mice were sacrificed when any tumors from a shared origin reached ∼3 cm3. (P) Comparison of RTVs and tumor weights at the end of the study between vehicle-treated (n = 22) and everolimus-treated (n = 22) mice. (Q) Heat maps showing protein (prot1 and prot2) and phosphopeptide (phos1 and phos2) signatures (rows) defining Sub1 and Sub2. The numbers of proteins and phosphopeptides are indicated in parentheses. (R) Box plots showing ratios of RTVs (left panel) and tumor weights (right panel) at endpoints in everolimus-treated samples compared with their paired vehicle-treated samples in Sub1 and Sub2. **, P < 0.01; *, P < 0.05 from two-sample t-test. (S) Cellular pathways significantly enriched by proteins (prot1 and 2) and phosphoproteins of selected phosphopeptides (phos1 and phos2) defining Sub1 and Sub2. The heat map shows pathway enrichment significance, represented as z-scores computed as -N−1(P-value), where P-value is the enrichment P-value from DAVID or ConsensusPathDB, and N−1(P-value) is the inverse normal distribution. (T) Network model showing interactions between proteins and phosphorylated proteins involved in leukotriene metabolic process (top), actin cytoskeleton regulation (bottom left), and neutrophil extracellular trap formation (NETosis, bottom right). Pink nodes indicate proteins (prot2) defining Sub2, while circled P on a node indicates phosphoproteins containing the phosphopeptides (phos2) defining Sub2. Solid arrows indicate direct activation; dotted arrows indicate indirect activation; arrows with "+p" denote phosphorylation. (U) Representative immunohistochemistry images of ALOX5 for the indicated responder and non-responder. Scale bar, 50 µm. Abbreviations: AA, arachidonic acid; DAG, diacylglycerol; PI, phosphatidylinositol. To determine whether mTOR inhibition also suppresses the progression of BRCA1-associated breast cancer, we tested the efficacy of everolimus on spontaneously developed mammary tumors in Brca1co/coMMTV-Cre mice through periodic observation and palpation. Tumor-bearing mice (size < 0.5 cm3) were then randomized to receive either vehicle or everolimus via oral gavage (Top right of Figure 1A, Supplementary Figure S4). Tumor volumes at baseline and during progression were measured weekly using magnetic resonance imaging (MRI) until the tumors reached a volume of ∼3 cm3 (Figure 1I). Tumors in vehicle-treated mice grew more rapidly than those in everolimus-treated mice (Figure 1J, left panel). An analysis of weekly progression showed that 73% (28 of 38) of tumors in vehicle-treated mice exhibited greater than 50% progression, compared to only 33% (46 of 141) in everolimus-treated mice (Figure 1J, right panel; P = 0.0002, chi-square test). Additionally, the weekly increase in tumor volume in the everolimus-treated group (51.0%, 95% CI: 40.3%-61.8%) was significantly lower (P < 0.001) than that in the vehicle-treated group (89.1%, 95% CI: 70.4%-107.7%). Moreover, everolimus-treated mice showed significantly longer survival (2.8-fold on average) compared to vehicle-treated mice (Figure 1K; P < 0.001). Importantly, while everolimus treatment significantly improved therapeutic outcomes in Brca1-mutant tumors, responses to everolimus showed heterogeneity among individual mice. Specifically, 8 of the 15 mice, designated as responder mice, exhibited a significant reduction in the ratio of tumor volume (RTV) in response to everolimus. In contrast, the remaining 7 mice, designated as non-responders, displayed a higher RTV than responders and vehicle-treated mice (Figure 1L,M). Additionally, the survival of responders (13.0 weeks) was nearly double that of non-responders (6.6 weeks) (Figure 1N). To further examine the therapeutic efficacy of everolimus, we employed an engraft model for preclinical evaluation. Tumor tissues were collected from 22 individual spontaneously developed mammary tumors in Brca1co/coMMTV-Cre mice, orthotopically transplanted into nude female mice, amplified, re-transplanted, and subsequently treated with either vehicle or everolimus. Tumor progression was monitored (bottom right of Figure 1A, Supplementary Figure S5), and all mice were sacrificed when any tumors in either vehicle- or everolimus-treated group reached ∼3 cm3 (Figure 1O, Supplementary Figure S6). Tumors from everolimus-treated mice showed significant reductions in RTV (43%) and weight (38%) compared to tumors from vehicle-treated mice (Figure 1P, Supplementary Figure S5). These findings suggest effective management of BRCA1-associated breast cancer by pharmacological mTOR inhibition. To explore this heterogeneity, we conducted global proteome and phosphoproteome profiling of vehicle- and everolimus-treated allograft tumors (Supplementary Figure S7A). Two distinct sample clusters (Sub1 and Sub2) were identified using both protein and phosphopeptide data (Supplementary Figure S7B,C). We identified 304 and 323 proteins that were upregulated, and 251 and 291 phosphopeptides that were upregulated, in Sub1 and Sub2, respectively (Figure 1Q). Sub2, characterized by higher RTVs and weights, represented the non-responders, whereas Sub1 corresponded to the responders (Figure 1R). The upregulated proteins and phosphoproteins in Sub2 were associated with neutrophil extracellular trap formation (NETosis) and leukotriene metabolism (Figure 1S). Enzymes involved in phosphatidylinositol and arachidonic acid formation/metabolism were upregulated in non-responders, leading to the release of leukotrienes (Figure 1T). Upon leukotriene binding, (1) proteins and phosphorylations mediating actin polymerization required for neutrophil migration, and (2) proteins involved in NETosis, were upregulated in non-responders (Figure 1T). Western blotting and immunohistochemistry confirmed the upregulation of representative markers of the leukotriene and NETosis pathways (Figure 1U, Supplementary Figure S7D,E). Therefore, our findings provide preclinical evidence that targeting mTOR inhibition is a potential strategy for the prevention and therapeutic management of BRCA1-associated breast cancer. Additionally, activation of the leukotriene-neutrophil activation axis can serve as a predictive marker of resistance to targeted mTOR inhibition. We further discussed the leukotriene signaling as a predictive biomarker and potential clinical translational value of this study in supplementary information. Chang-il Hwang, Daehee Hwang, and Sang Soo Kim conceived the study, designed, and supervised the experiments. Tae Hyun Kim, Chu-Xia Deng, Sung Chul Lim, Chang-il Hwang, Daehee Hwang, and Sang Soo Kim wrote and revised the manuscript. Eun Jung Park, Tae Hyun Kim, Dong Hoon Shin, Heesun Cheong, Chu-Xia Deng, Sung Chul Lim, Chang-il Hwang, Daehee Hwang, and Sang Soo Kim contributed to the data analysis and interpretation. Hye Jung Baek, Jihao Xu, and Heesun Cheong contributed to the in vitro experiments. Eun Joo Cho, Min Kyung Ki, and Dong Hoon Shin conducted the in vivo animal experiments. Geun Hee Han performed bioinformatical analysis. Tae Hyun Kim executed the statiscal analysis. Sung Chul Lim performed the pathological analysis. Eun Jung Park performed immunological analysis. All authors read and approved the final manuscript. We would like to thank Core facilities at National Cancer Center Korea for supporting analysis. The authors declare no conflict of interest. This work was supported by the National Cancer Center of Korea (NCC-2210680/2410880) and the National Research Foundation of Korea (2023R1A2C1004000). All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of the National Cancer Center of Korea (NCC-15-295). All generated global and phosphoproteomic data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository, with PXD054301 as the identifier [10]. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

We report proteogenomic analysis of locally advanced cervical cancer (LACC). Exome-seq data revealed predominant alterations of keratinization-TP53 regulation and O-glycosylation-TP53 regulation axes in squamous and adeno-LACC, respectively, compared to in early-stage cervical cancer. Integrated clustering of mRNA, protein, and phosphorylation data identified six subtypes (Sub1-6) of LACC among which Sub3, 5, and 6 showed the treatment-resistant nature with poor local recurrence-free survival. Elevated immune and extracellular matrix (ECM) activation mediated by activated stroma (PDGFD and CXCL1high fibroblasts) characterized the immune-hot Sub3 enriched with MUC5AChigh epithelial cells (ECs). Increased epithelial-mesenchymal-transition (EMT) and ECM remodeling characterized the immune-cold squamous Sub5 enriched with PGK1 and CXCL10high ECs. We further demonstrated that CIC mutations could trigger EMT activation by upregulating ETV4, and the elevation of the immune checkpoint PVR and neutrophil-like myeloid-derived suppressive cells (FCN1 and FCGR3Bhigh macrophages) could cause suppression of T-cell activation in Sub5. Increased O-linked glycosylation of mucin characterized adeno-LACC Sub6 enriched with MUC5AChigh ECs. These results provide a battery of somatic mutations, cellular pathways, and cellular players that can be used to predict treatment-resistant LACC subtypes and can serve as potential therapeutic targets for these LACC subtypes.

Liver toxicity poses a critical challenge in drug development due to the liver's pivotal role in drug metabolism and detoxification. Accurately predicting liver toxicity is crucial but is hindered by scattered information sources, a lack of curation standards, and the heterogeneity of data perspectives. To address these challenges, we developed the HepatoToxicity Portal (HTP), which integrates an expert-curated knowledgebase (HTP-KB) and a state-of-the-art machine learning model for toxicity prediction (HTP-Pred). The HTP-KB consolidates hepatotoxicity data from nine major databases, carefully reviewed by hepatotoxicity experts and categorized into three levels: in vitro, in vivo, and clinical, using the Medical Dictionary for Regulatory Activities (MedDRA) terminology. The knowledgebase includes information on 8,306 chemicals. This curated dataset was used to build a hepatotoxicity prediction module by fine-tuning a GNN-based foundation model, which was pre-trained with approximately 10 million chemicals in the PubChem database. Our model demonstrated excellent performance, achieving an area under the ROC curve (AUROC) of 0.761, surpassing existing methods for hepatotoxicity prediction. The HTP is publicly accessible at https://kobic.re.kr/htp/ , offering both curated data and prediction services through an intuitive interface, thus effectively supporting drug development efforts. Scientific contributions HTP-KB consolidates comprehensive curated information on liver toxicity gathered from nine sources. HTP-Pred utilizes advanced deep learning techniques, significantly enhancing predictive accuracy. Together, these tools provide valuable resources for researchers and practitioners in drug development, accessible through a user-friendly interface.

Naïve CD4 T cells are traditionally viewed as a quiescent, homogeneous, resting population, but emerging evidence reveals their heterogeneity, which can be crucial for understanding disease contexts and therapeutic outcomes. In this study, we identify distinct subpopulations within both murine and human naïve CD4 T cells by single cell-RNA-sequencing (scRNA-seq), particularly focusing on a subpopulation that expresses super-high levels of interleukin-7 receptor (IL-7Rsup-hi), along with CD97, IL-18R, and Ly6C. This subpopulation, absent in the thymus and peripherally induced, exhibits type 1 helper T cell (Th1)-poised characteristics and contributes to the inhibition of cancer progression in B16F10 tumor-bearing mice. In humans, this IL-7Rsup-hi subpopulation expressing CD97 correlates with the responsiveness to anti-PD-1 therapy in cancer patients and the disease state of multiple sclerosis. By elucidating the heterogeneity of naive CD4 T cells and identifying a Th1-poised subpopulation capable of robust type 1 responses, we highlight the importance of this heterogeneity in inflammatory conditions for defining the disease states and predicting drug responsiveness. The naïve CD4 T cell compartment is traditionally deemed as homogenous. Here, by leveraging scRNA-seq analysis, the authors reveal the role of naive CD4 T cell heterogeneity in disease states and therapeutic outcomes by characterizing a Th1-poised naive CD4 T cell subpopulation, linked to anti-tumor immunity in mice and responsiveness to anti-PD-1 therapy in humans.

Retinol-binding protein 4 (RBP4), the sole specific carrier for retinol (vitamin A) in circulation, is highly expressed in liver and adipose tissues. Previous studies have demonstrated that RBP4 plays a role in cold-mediated adipose tissue browning and thermogenesis. However, the role of RBP4 in brown adipose tissue and its metabolic significance remain unclear. Here we generated and studied transgenic mice that express human RBP4 (hRBP4), specifically in brown adipocytes (UCP1-RBP4 mice), to better understand these uncertainties. When fed a chow diet, these mice presented significantly lower body weights and fat mass than their littermate controls. The UCP1-RBP4 mice also showed significant improvements in glucose clearance, enhanced energy expenditure and increased thermogenesis in response to a cold challenge. This was associated with increased lipolysis and fatty acid oxidation in brown adipose tissue, which was attributed to the activation of canonical adrenergic signaling pathways. In addition, high-performance liquid chromatography analysis revealed that plasma RBP4 and retinol levels were elevated in the UCP1-RBP4 mice, whereas their hepatic retinol levels decreased in parallel with a chow diet. Steady-state brown fat levels of total retinol were significantly elevated in the UCP1-RBP4 mice, suggesting that their retinol uptake was increased in RBP4-expressing brown adipocytes when fed a chow diet. These findings reveal a critical role for RBP4 in canonical adrenergic signaling that promotes lipid mobilization and oxidation in brown adipocytes, where the harnessed energy is dissipated as heat by adaptive thermogenesis.

Airspace or aerenchyma is crucial for plant development and acclimation to stresses such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation in stems and roots under hypoxic conditions, the precise mechanisms remain unclear. Moreover, the cellular dynamics underlying airspace formation in shoots are poorly understood. We investigated the stage-dependent structural dynamics of shoot aerenchyma in greater duckweed (Spirodela polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we showed that the spatial framework of aerenchyma is established before frond volume increases, driven by cell division and expansion. The substomatal cavity connecting aerenchyma to stomata formed via programmed cell death (PCD) and was closely associated with guard cell development. Additionally, transcriptome analysis and pharmacological studies revealed that the organization of aerenchyma in greater duckweed is determined by the interplay between PCD and proliferation. This balance is governed by spatiotemporal regulation of phytohormone signaling involving ethylene, abscisic acid, and salicylic acid. Overall, our study reveals the structural dynamics and phytohormonal regulation underlying aerenchyma development in duckweed, improving our understanding of how plants establish distinct architectural arrangements. These insights hold the potential for wide-ranging application, not only in comprehending aerenchyma formation across various plant species but also in understanding how airspaces are formed within the leaves of terrestrial plants.

Background In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. Methods To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. Results CD244 fl/fl LysM cre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244 fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244 fl/fl LysM cre mice also showed higher percentages of Ly6C low macrophages, along with elevated gp100 + IFN-γ + CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6C low macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244 −/− bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Conclusion Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Vernalization and cold acclimation are plant strategies that evolved to enhance their fitness during the winter season. The molecular mechanisms behind these processes in Arabidopsis have been intensively studied. However, how plants measure the duration of long-term cold exposure has not been fully understood. Our research shows that cytosolic chaperonin is crucial for appropriate long-term cold responses by regulating plant circadian clocks. Furthermore, one of the clock components, REVEILLE4 (RVE4) and RVE8 directly activate VERNALIZATION INSENSITIVE 3 ( VIN3 ) and the C-REPEAT BINDING FACTOR ( CBF )/ DEHYDRATION-RESPONSIVE ELEMENT BINDING 1 ( DREB1 ) family genes, which are key regulators of vernalization and cold acclimation, respectively. The activation of the VIN3 and CBF/DREB1 genes was specific to the time of day, indicating that cold exposure during the day is critical for long-term cold responses. Our research delves deeper into understanding the regulatory mechanism governing these two distinct long-term cold responses.

Background: Poor clinical outcomes and mortality among those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to a hyperinflammatory state. We studied the correlation between a wide variety of COVID-19 clinical outcomes and viral load, Nucleocapsid (NP), NP-specific IgM and IgG, cytokine levels, and macrophage activation.Methods: We compared the levels of SARS-CoV-2 viral load, NP, NP-specific IgM and IgG titers, and cytokine levels among those that experienced asymptomatic, mild, moderate and critical/severe disease. Using in vitro studies, we evaluated the effect of THP‐1-derived macrophages that were treated with NP and NP-IgG monoclonal antibodies on select cytokines including IL-6, IL-10, and TGF‐β both before and after IL-10 inhibition.Findings: Viral load in severe/critical disease patients was not significantly different from that in asymptomatic patients. Elevated levels of NP specific IgG, IL-6, and IL-10 as well as low levels of NP and TGF-β were linked to disease severity and high levels of IFN-γ are also associated with mortality in COVID-19 patients. In vitro studies showed that NP with NP-specific IgG can induce M2b macrophages (HLA-DRHighCCR7-CD86+), which contributed the hyperproduction of IL-1β, IL-6, IL-10, and TNF-α and low production of TGF‐β. Finally, inhibition of IL‐10 signaling decreased the production of IL‐6, IL-10, and TNF-α and elevated that of TGF‐β.Interpretation: NP with NP-specific IgG can induce M2b macrophages (HLA-DRHighCCR7-CD86+) and can induce the hyperproduction of IL-6 and IL-10 and low production of TGF‐β leading to worse clinical outcomes in COVID-19 patients. IL-10 likely plays an important role in the host immune response to severe and critical SARS-CoV-2 infection and could serve as a prognostic or therapeutic target.Funding: This study was supported by grants from the National Research Foundation of Korea (NRF) and the Ministry of Science, ICT, and Future Planning (grant number: NRF-2021R1A2C2091578) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which is funded by the National Institute of Infectious Diseases, National Institute of Health, Republic of Korea (grant number: HD22C2045).Declaration of Interest: The authors declare no competing interests.Ethical Approval: The study was approved by the Institutional Review Board (IRB) at Jeju National University Hospital (IRB file no. 2020-10-019).