Search Results (246)

The incorporation of nucleoside analogs into DNA by polymerases, followed by their removal through base excision repair (BER), represents a promising strategy for cancer chemotherapy. In this study, we investigated the incorporation and cytotoxic effects of several nucleoside analogs—some of which are epigenetic reprogramming intermediates—in the U87 glioblastoma cell line. We found that two analogs, 5-hydroxymethyl-2′-deoxyuridine (5HmdU) and trifluorothymidine (TFT), are both cytotoxic and are efficiently incorporated into genomic DNA. In contrast, the 5-carboxy analogs—5-carboxy-2′-deoxyuridine (5CadU) and 5-carboxycytidine (5CadC)—showed no cytotoxicity and were not incorporated into DNA. Interestingly, 5-hydroxymethyl-2′-deoxycytidine (5HmdC) was cytotoxic but was not directly incorporated into DNA. Instead, it was deaminated into 5HmdU, which was then incorporated and likely responsible for the observed toxicity. 5HmdU is actively removed from DNA through the BER pathways. In contrast, TFT remains stably incorporated and is neither excised by BER nor does it hydrolyze into 5CadU—a known substrate for the DNA glycosylase SMUG1. We also found that N⁶-benzyladenosine (BzAdo), an inhibitor of the enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase (DNPH1), enhances the cytotoxicity of 5HmdU. However, the thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI) does not increase the cytotoxic effect of TFT in U87 cells. Together, these findings highlight 5HmdU and TFT as promising chemotherapeutic agents for glioblastoma, each with distinct mechanisms of action and cellular processing. Full article

Murine microglia exhibit rapid self-renewal upon removal from the postnatal brain. However, the signaling pathways that regulate microglial repopulation remain largely unclear. To address this knowledge gap, we depleted microglia from mixed glial cultures using anti-CD11b magnetic particles and cultured them for 4 weeks to monitor their repopulation ability in vitro. Flow cytometry and immunocytochemistry revealed that anti-CD11b bead treatment effectively eliminated >95% of microglia in mixed glial cultures. Following removal, the number of CX3CR1-positive microglia gradually increased; when a specific threshold was reached, repopulation ceased without any discernable rise in cell death. Cell cycle and 5-ethynyl-2′-deoxyuridine incorporation assays suggested the active proliferation of repopulating microglia at d7. Time-lapse imaging demonstrated post-removal division of microglia. Colony-stimulating factor 1 receptor-phosphoinositide 3-kinase-protein kinase B signaling was identified as crucial for microglial repopulation, as pharmacological inhibition or neutralization of the pathway significantly abrogated repopulation. Transwell cocultures revealed that resident microglia competitively inhibited microglial proliferation probably through contact inhibition. This in vitro microglial removal system provides valuable insights into the mechanisms underlying microglial proliferation. Full article

Background: Esophageal squamous cell carcinoma (ESCC) is a fatal malignant tumor. Several studies have demonstrated that immune checkpoint inhibitors can provide clinical benefits to patients with ESCC. However, the single-agent efficacy of these agents remains limited. Although combination therapies (e.g., radiotherapy, chemotherapy) can help to overcome immunotherapy resistance in ESCC, their severe side effects limit clinical application. This study aimed to explore new resistance mechanisms to immunotherapy in ESCC and identify novel molecular targets to overcome immunotherapy resistance. Methods: We employed immunohistochemistry staining to examine the p-FGFR1^Y654 in tumor samples obtained from 103 patients with ESCC, in addition to evaluating CD8+ T cell infiltration. In vitro expression, western blotting, CCK-8, 5-bromo-2′-deoxyuridine incorporation assays, and migration assays were used to confirm the impact of AZD4547 on p-FGFR1^Y654 expression and the proliferation and migration in ESCC cell lines. Through RNA sequencing analysis, databases such as the Cancer Genome Atlas (TCGA) and Gene Set Cancer Analysis (GSCA), and the reconstruction of transgenic mice using the humanized immune system, we validated the correlation between the expression of p-FGFR1^Y654 and CD8+ T cell infiltration. We also explored how p-FGFR1^Y654 recruits myeloid-derived suppressor cells (MDSCs) through the CXCL8–CXCR2 axis to suppress the therapeutic efficacy of immunotherapy in ESCC. Finally, the tumor-suppressive effects of AZD4547 combined with immunotherapy were confirmed in vivo in tumor-bearing mice with a humanized immune system. Results: We found that the inhibition of p-FGFR1^Y654 expression in ESCC can enhance CD8+ T cell infiltration by suppressing the CXCL8-–XCR2 recruitment of MDSCs. AZD4547, combined with immunotherapy, further promotes immunotherapeutic efficacy in ESCC. Conclusions: In conclusion, our study presents a promising model for combination therapy in ESCC immunotherapy. Full article

Marine tunicates are a very attractive and abundant source of secondary metabolites with chemical diversity and biological activity. Fractionation and purification of the organic extract of the Red Sea tunicate Didemnum species resulted in the isolation and identification of three new compounds, didemnosides A and B (1 and 2) and 1,1′,3,3′-bisuracil (3), together with thymidine (4), 2′-deoxyuridine (5), homarine (6), and acetamide (7). Planar structures of the compounds were explained through analyses of their 1D (¹H and ¹³C) and 2D (¹H–¹H COSY, HSQC, and HMBC) NMR spectra and high-resolution mass spectral determinations. Compound 1 exhibited the highest growth inhibition toward the MCF-7 cancer cell line with IC₅₀ values of 0.597 μM, while other compounds were inactive (≥50 μM) against this cell line. On the other hand, compounds 1, 2, and 4–7 moderately inhibited SW-1222 and PC-3 cells with IC₅₀ values ranging between 5.25 and 9.36 μM. Molecular docking analyses of the top three active compounds on each tested cell line exposed stable interactions into the active pockets of estrogen receptor alpha (ESR1), human topoisomerase II alpha (TOP2A), and cyclin-dependent kinase 5 (CDK5) which are contemplated as essential targets in cancer treatments. Thus, compound 1 represents a scaffold for the development of more effective anticancer drugs. Full article

Mutations in the TP63 gene cause several syndromic disorders, including ankyloblepharon–ectodermal defects–cleft lip/palate (AEC) syndrome, characterized by severe skin erosions, cleft palate, and ectodermal dysplasia. These mutations often affect the carboxy-terminal sterile-α-motif (SAM) domain of the p63 protein, leading to domain misfolding, protein aggregation, and impaired transcriptional activity. To dissect the molecular mechanisms underlying AEC pathogenesis, we investigated primary keratinocytes derived from p63L514F mutant mice, which carry a SAM domain mutation associated with AEC syndrome. p63L514F keratinocytes exhibited significantly reduced proliferation compared to wild-type controls, as indicated by decreased 5-ethynyl-2′-deoxyuridine (EdU) incorporation, decreased Cyclin D1 and Cyclin D2 expression, and an increase in the cell-cycle inhibitors p21 and p27. Furthermore, p63L514F keratinocytes showed increased cell death, elevated reactive oxygen species (ROS) levels, and a decreased reduced (GSH) and oxidized (GSSG) glutathione (GSH/GSSG) ratio, indicating oxidative stress. This stress response was accompanied by a marked reduction in Solute Carrier Family 7 Member 11 (Slc7a11), a critical regulator of antioxidant defense. We further identified Slc7a11 as a likely direct transcriptional target of p63: p63 depletion reduced Slc7a11 expression, and chromatin immunoprecipitation uncovered an evolutionary conserved p63-binding enhancer upstream of the Slc7a11 promoter. Together, our findings demonstrate that p63 mutations causative of AEC syndrome impair keratinocyte proliferation, promote cell death via oxidative stress, and compromised antioxidant defenses, revealing a dual role for p63 in sustaining skin homeostasis. Full article

Bacteria are ubiquitous in the environment and critical to human health and disease, yet only a small fraction can be identified through standard culture methods. Advances in next-generation sequencing techniques have improved bacterial identification, but these DNA-based methods cannot distinguish live bacteria from relic DNA. Recently, DNA-labeling dyes (e.g., 5-bromo-2′-deoxyuridine [BrdU] and propidium monoazide [PMA]) have been used to detect metabolically active bacteria in different sample types. Here, we compare BrdU and PMA in combination with 16SrRNA gene sequencing to characterize metabolically active bacteria in two different sample types: (1) manufactured products (n = 78; cigarettes, hookah, and little cigar) and (2) natural samples (n = 186; rainwater, soil, and produce). Metabolically active bacterial communities identified in BrdU-labeled samples had lower alpha diversity than that of PMA-treated and non-treated samples. Pseudomonas, Sphingomonas, Enterobacter, and Acinetobacter were observed in all the samples tested. Irrespective of sample type, Pseudomonas was predominant in BrdU-treated samples, while Acinetobacter was more abundant in non-treated samples compared to PMA-treated samples. We also observed that PMA-treated samples tend to overestimate the metabolically active bacterial fraction compared to BrdU-treated samples. Overall, our study highlights how different labeling techniques influence bacterial community analysis findings, underscoring the need for careful selection of labeling approaches when assessing environmental samples. Full article

The purpose of this study was to examine the effect of daidzein on the proliferation of porcine mammary epithelial cells (PMECs) and elucidate the underlying molecular mechanisms. PMECs were treated with varying daidzein or rapamycin levels, and then cell proliferation and mTOR pathway protein expression were detected. When the concentration of daidzein added was in the range of 0–80 μM, cell proliferation was significantly promoted (p < 0.05). These results were in agreement with those obtained using the 5-ethynyl-2′-deoxyuridine (EdU) assay. Daidzein administration at 20 and 40 μM concentrations triggered significant activation of the mTOR signaling cascade and enhanced expression of downstream cell-cycle-regulatory proteins (cyclin D1) (p < 0.05). Moreover, exposure to 40 μM daidzein attenuated apoptotic signaling, as evidenced by reduced levels of Bax protein and cleaved caspase-3 (p < 0.05). These effects were reversed when rapamycin was used to inhibit the mTOR pathway. In conclusion, our findings suggest that daidzein activates PMEC proliferation via the mTOR pathway. The present work not only characterizes new functional properties of daidzein but also establishes mechanistic evidence supporting its role in augmenting sow lactation efficiency. Full article

The DHRS3 gene, a member of the short-chain dehydrogenase/reductase (SDR) family, is involved in critical metabolic processes in animals. This study investigated the expression patterns of DHRS3 across various tissues of developmental stages in pigs and preliminarily evaluated its effects on myoblast proliferation, apoptosis, and differentiation. RT-qPCR (real-time quantitative PCR) was employed to analyze DHRS3 expression in the heart, liver, spleen, lungs, kidneys, longissimus dorsi, foreleg, and hind leg of pigs at 3 days, 6 months, and 12 months of age. Cell proliferation was analyzed using EdU (5-ethynyl-2′-deoxyuridine) assays, RT-qPCR, and flow cytometry, while the expression changes of proliferation-, apoptosis-, and differentiation-related genes were assessed via RT-qPCR. The results indicated that DHRS3 was expressed in all eight tissues at all three developmental stages. At 3 days, DHRS3 expression was the highest in the kidneys; at 6 months, it peaked in the liver; and at 12 months, it was again the highest in the kidneys. Across all stages, the liver and kidneys exhibited the highest DHRS3 expression levels. Functional studies revealed that DHRS3 overexpression suppressed myoblast proliferation and differentiation while promoting apoptosis. In contrast, DHRS3 inhibition enhanced myoblast proliferation and differentiation and reduced apoptosis. These findings underscore the regulatory role of DHRS3 in myogenesis and provide insights into its metabolic and developmental functions in pigs. Full article

Amomum villosum L. is a perennial herbaceous belonging to the ginger family. Due to its unique aroma, it is widely used in alcoholic beverages and food processing. Unfortunately, issues with bitterness and sourness occur, which affect the taste and quality of processed products. In this study, the non-volatile sour and bitter substances in Amomum villosum L. were systematically isolated, purified, and characterized through a combination of chromatographic separation techniques and ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The results indicate that three sour compounds (DL-malic acid, protocatechuic acid, and p-hydroxybenzoic acid) and one bitter compound (catechin) were identified for the first time in Amomum villosum L. The in vitro anti-tumor activity was screened and determined using Cell Counting Kit-8 (CCK-8) assays, a 5-Ethynyl-2′-deoxyuridine (EdU) staining experiment, and scratch assays. The results reveal that the bitter substance of catechin (25–100 μg/mL) exhibited significant inhibitory effects, which inhibited the proliferation and migration of human non-small cell lung cancer A549 cells through dose-dependent mechanisms. This investigation also reveals the influence of different traditional extraction solvents on the degree of bitterness and sourness in Amomum villosum extracts, providing a theoretical basis for improving the quality and pharmacological utilization of Amomum villosum extracts. Full article

Introduction: Non-small cell lung cancer (NSCLC) is a lethal type of lung cancer (LC) with a 5-year survival rate of 19%. Because drug resistance typically develops following chemotherapy, radiotherapy, and immunotherapy, a novel NSCLC therapeutic strategy is urgently demanded. Gambogenic acid (GNA), a major bioactive ingredient isolated from gamboge, has multipotent antitumor effects, although activity against NSCLC is unknown. Methods: CCK8, ethynyl deoxyuridine (EdU), the plate colony formation assay, and the transwell and wound healing (WH) assay were used to study the effect of GNA on the proliferation and migration ability of NSCLC. Flow cytometry was used to detect apoptosis and the cell cycle. Proteomic analysis and LiP-SMap were used to detect the downstream target of GNA. Ferroptosis inhibitor ferrostatin-1 was used to detect the effect of GNA on NSCLC ferroptosis. Overexpressing GCH1 was used for a rescue experiment. Subcutaneous tumor and pulmonary metastasis in a mouse model were used to study the effect of GNA on NSCLC growth and metastasis. Results: The results of the present study showed that GNA inhibited the proliferation and migration of NSCLC cells in a dose- and time-dependent manner, which arrested the cell cycle in the G0/G1 phase. In vivo data revealed that GNA inhibited tumor growth and lung metastasis. Proteomic analysis found that GNA significantly inhibited the expression of GTP cyclohydrolase 1 (GCH1). LiP-SMap analysis showed that GNA interacted with ILE248 and ARG249 of GCH1. GCH1 overexpression had a similar role to the ferroptosis inhibitor ferrostatin-1 and restored cell proliferation and migration after GNA treatment. Also, GNA promoted reactive oxygen species (ROS) accumulation, which reduced mitochondrial membrane potential. GCH1 overexpression or ferrostatin-1 treatment reversed GNA regulation of ROS accumulation and mitochondrial membrane potential inhibition. Conclusions: Taken together, these findings confirmed that GNA suppressed the malignant progression of NSCLC by inducing GCH1-mediated ferroptosis. Full article

2′-Deoxythymidine-5′-monophosphate, dTMP, is an essential precursor of thymine, one of the four canonical bases of DNA. In almost all living organisms, dTMP is synthesized de novo by a reductive methylation reaction of 2′-deoxyuridine-5′-monophosphate (dUMP) catalyzed by the thymidylate synthase, where the carbon used for the methylation is derived from methylenetetrahydrofolate (CH2THF). Many microbes, including human pathogens, utilize the flavin-dependent thymidylate synthase encoded by the thyX gene to generate dTMP. The mechanism of action relies on the reduced coenzyme FADH⁻, which acts both as a mediator, facilitating methylene transfer from CH2THF to dUMP, and as a reducing agent. Here, we present for the first-time crystallographic structures of ThyX from Thermotoga maritima in the reduced state alone and in complex with dUMP. ThyX flavin reduction appears to order the active site, favoring a flavin conformation that drastically deviates from that observed in the oxidized enzyme. The structures show that FADH⁻ potentially controls access to the folate site and the conformation of two active site loops, affecting the degree of accessibility of substrate pockets to the solvent. Our results provide the molecular basis for the sequential enzyme mechanism implemented by ThyX during dTMP biosynthesis. Full article

The Spodoptera frugiperda Sf9 insect cell line is used in the baculovirus expression vector system for the development of various viral vaccines and some gene therapy products. Early studies indicated that Sf9 cells produced a reverse transcriptase (RT) activity that was detected using a sensitive PCR-enhanced reverse transcriptase (PERT) assay. Since RT is generally associated with retrovirus particles, we undertook the investigation of the physical properties and infectious nature of the extracellular RT activity that was constitutively expressed from Sf9 cells or induced after the chemical treatment of the cells with drugs known to activate endogenous retroviruses. A density gradient analysis indicated that the peak RT activity corresponded to a low buoyant density of about 1.08 g/mL. Ultracentrifugation and size filtration of cell-free Sf9 supernatant indicated that different particle sizes were associated with the RT activity. This was confirmed by transmission electron microscopy and cryoEM, which revealed a diversity in particle size and type, including viral-like and extracellular vesicles. The treatment of Sf9 cells with 5-iodo-2′-deoxyuridine (IUdR) induced a 33-fold higher RT activity with a similar low buoyant density compared to untreated cells. Infectivity studies using various target cells (human A204, A549, MRC-5, and Raji, and African green monkey Vero cells) inoculated with cell-free supernatant from untreated and IUdR-treated Sf9 cells showed the absence of a replicating retrovirus by PERT-testing of cell-free supernatant during the 30 day-culturing period. Additionally, there was no evidence of virus entry by whole genome analysis of inoculated MRC-5 cells using high-throughput sequencing. This is the first study to identify extracellular retroviral-like particles in Spodoptera. Full article

Background. The use of the bone-seeking properties of bisphosphonates (BPs) to target the delivery of therapeutic drugs is a promising approach for the treatment of bone metastases. Currently, the most advanced example of this approach is a gemcitabine-ibandronate conjugate (GEM-IB), where the bone-targeting BP ibandronate (IB) is covalently linked to the antineoplastic agent gemcitabine (GEM) via a spacer phosphate group. In the present study, we describe the development of a new analytical platform to evaluate the metabolism and pharmacokinetics of GEM-IB in mice and dogs and the results of proof-of-concept studies assessing the pharmacokinetics of GEM-IB in dogs and mice. Methods. We validated analytical platforms to analyze GEM-IB and five of its major metabolites IB, gemcitabine-5′-phosphate (GEMMP), gemcitabine (GEM), 2′,2′-difluoro-2′-deoxyuridine-5′-phosphate (dFdUMP), and 2′,2′-difluoro-2′-deoxyuridine (dFdU) and performed proof-of-concept pharmacokinetic studies in mice (5 mg/kg i.p.) and dogs (5 mg/kg i.v.). Results. Intra- and inter-run accuracy and imprecision (3 days) of the assays met the (FDA) acceptance criteria. The proof-of-concept plasma pharmacokinetic studies in mice showed AUCs of 1278, 10,652, 405, 38, 1063, 3389, and 38 h·ng/mL for GEM-IB, IB, GEMMP, dFdU-MP, GEM, and dFdU, respectively. In dog plasma, AUCs of 295, 5725, 83, 11, 1625, and 6569 h·ng/mL were observed for GEM-IB, IB, GEMMP, dFdUMP, GEM, and dFdU. Conclusions. Pharmacokinetic studies in dogs and mice showed that GEM-IB is rapidly converted to IB and GEM; dFdU is formed (from GEM) with a delay. The rapid disappearance of GEM-IB from circulation could be explained by a combination of metabolism and rapid distribution to tissue/bone. Full article

Traumatic brain injury (TBI) is one of the major causes of severe neurological disorders and long-term dysfunction in the nervous system. Besides inducing neurodegeneration, TBI alters stem cell activity and neurogenesis within primary neurogenic niches. However, the fate of dividing cells in other brain regions remains unclear despite offering potential targets for therapeutic intervention. Here, we investigated cell division and differentiation in non-neurogenic brain regions during the acute and delayed phases of TBI-induced neurodegeneration. We subjected mice to lateral fluid percussion injury (LFPI) to model TBI and analyzed them 1 or 7 weeks later. To assess cellular proliferation and differentiation, we administered 5-ethinyl-2′-deoxyuridine (EdU) and determined the number and identity of dividing cells 2 h later using markers of neuronal precursors and astro-, micro-, and oligodendroglia. Our results demonstrated a significant proliferative response in several brain regions at one week post-injury that notably diminished by seven weeks, except in the optic tract. In addition to active astro- and microgliosis, we detected oligodendrogenesis in the striatum and optic tract. Furthermore, we observed trauma-induced neurogenesis in the striatum. These findings suggest that subcortical structures, particularly the striatum and optic tract, may possess a potential for self-repair through neuronal regeneration and axon remyelination. Full article

Sex-controlled sperm combined with artificial insemination allows animals to reproduce offspring according to the desired sex, accelerates the process of animal genetics and breeding and promotes the development of animal husbandry. However, the molecular markers for sexual sperm sorting are unusual. To identify the molecular markers of boar sperm sorting, proteomics and metabolomics techniques were applied to analyze the differences in proteins and metabolism between X and Y sperm. Label-free quantitative proteomics identified 254 differentially expressed proteins (DEPs) in the X and Y sperm of boars, including 106 proteins that were highly expressed in X sperm and 148 proteins that were highly expressed in Y sperm. Among the differential proteins, COX6A1, COX1, CYTB, FUT8, GSTK1 and PFK1 were selected as potential biological markers for X and Y sperm sorting. Moreover, 760 metabolites from X and Y sperm were detected. There were 439 positive ion mode metabolites and 321 negative ion mode metabolites identified. The various metabolites were phosphoenolpyruvate, phytosphingosine, L-arginine, N-acetylputrescine, cytidine-5′-diphosphate and deoxyuridine. These metabolites were mainly involved in the TCA cycle, oxidative phosphorylation pathway, glycolysis pathway, lipid metabolism pathway, amino acid metabolism pathway, pentose phosphate pathway and nucleic acid metabolism pathway. The differential proteins and differential metabolites obtained by the combined proteomics and metabolomics analysis were projected simultaneously to the KEGG pathway, and a total of five pathways were enriched, namely oxidative phosphorylation pathway, purine metabolism, unsaturated fatty acid biosynthesis, ABC transporters and peroxisomes. In summary, COX6A1 and CYTB were identified as potential biomarkers for boar X and Y sperm sorting. Full article