Search Results (283)

KRAS is one of the most frequently mutated genes in all human cancers, and its oncogenic mutation hotspots are glycine 12 (G12), glycine 13 (G13), glutamine 61 (Q61) and alanine 146 (A146). Among these hotspot mutations, A146 substitution mutations (A146X) occur relatively infrequently, except for haematopoietic and lymphoid cancers, suggesting that A146X causes intrinsically distinct KRAS signalling compared to other KRAS oncogenic alleles. However, due to the absence of model A146X cell lines derived from haematopoietic sources, the cellular mechanisms that cause the differences between KRAS.A146X and other common KRAS mutants, such as KRAS.G12X, remain largely unexplored. In this study, we developed a set of isogenic model haematopoietic cell lines expressing KRAS.A146T, KRAS.G12V and KRAS.G12G (non-mutated) from the endogenous locus by genetically modifying the human lymphoblastoid TK6 cell line. We found that TK6 cells carrying KRAS^A146T/+ or KRAS^G12V/+ exhibited increased replication stress compared to KRAS wild-type cells. Strikingly, KRAS^A146T/+ cells strongly rely on PrimPol for maintaining cellular survival upon replication stress. In contrast, KRAS^G12V/+ cells exhibited hypersensitivity to inhibitors for the ATR-Chk1 checkpoint signalling axis and to nucleoside analogues commonly used to treat cancers and viral infections. Our findings demonstrate that the endogenously expressed oncogenic KRAS mutations exacerbate the replication stress and reveal KRAS allele-specific replication phenotypes, facilitating the development of effective chemotherapies tailored to specific oncogenic KRAS mutation alleles and types of cancer. Moreover, our study offers valuable model cell lines for investigating mechanisms underlying replication vulnerability in cancers harbouring oncogenic KRAS mutations. Full article

Flammulina filiformis is a widely cultivated edible mushroom valued for its taste and nutrition. However, its stipe often develops a fibrous and stringy texture that unpleasantly lodges between teeth during chewing. Texture analysis confirmed a distinct toughness gradient, with the upper stipe being more brittle and less tough than the lower part. UHPLC-MS/MS-based metabolomics of these regions identified 953 metabolites, predominantly spanning lipids and lipid-like molecules, organic acids and derivatives, and nucleosides, nucleotides, and analogues. Comparative analysis revealed that the tender upper stipe was characterized by a widespread downregulation of primary metabolites, including severe depletion of key signaling molecules (cAMP, cGMP) and amino acids such as L-tryptophan. In contrast, the tough lower stipe was enriched with metabolites indicative of an oxidative environment, notably a broad spectrum of oxidized lipids and phenolic compounds. KEGG pathway analysis attributed this dichotomy to distinct metabolic programs. While the upper stipe exhibited downregulation in tryptophan and purine metabolism, the lower stipe was enriched for pathways associated with redox homeostasis and lipid peroxidation, including glutathione metabolism and lipid peroxidation. The co-accumulation of oxidized lipids and phenolics suggests a potential mechanism for oxidation-driven tissue fortification. This study reveals a spatially programmed metabolic basis for the textural differentiation in F. filiformis stipes, providing a framework for understanding tissue development and highlighting potential regulatory targets for breeding varieties with improved eating quality. Full article

Nucleic acids are essential dietary components with diverse physiological functions. Numerous studies have focused on the biological functions of nucleotides, nucleosides, and functional RNAs such as microRNAs. However, the nutritional value of ribosomal RNA (rRNA)-derived oligonucleotides, which are likely the predominant nucleic acid-derived components in foods, remains largely unexplored. Here, yeast was used as a food-associated eukaryotic model organism to investigate the uptake and utilization of rRNA-derived oligonucleotides. Yeast efficiently utilized short RNA oligonucleotides (approximately 5–30 nt) as nutrient sources, supporting robust cell growth. Confocal microscopy confirmed rapid uptake of Cy5-labeled RNA oligonucleotides by yeast cells. Proteomic analysis further revealed marked upregulation of proteins involved in endocytosis and autophagy in yeast cultured with RNA oligonucleotides. Collectively, these findings demonstrate that yeast can internalize and metabolize rRNA-derived oligonucleotides as efficient nutrient sources, likely through coordinated endocytic and autophagic pathways. This study highlights the nutritional potential of rRNA-derived oligonucleotides and provides a foundation for their future application in functional foods and fermentation systems. Full article

Background: Infections caused by the protozoan Trichomonas vaginalis affect millions of people worldwide and are responsible for one of the most common sexually transmitted diseases. Despite the efficacy of 5-nitroimidazoles like metronidazole, concerns regarding widespread resistance and the absence of viable alternatives for specific patient populations necessitate the development of structurally diverse pharmacological agents. In this study, we investigated the antiparasitic activity of 1,3-thiazolidin-4-one derivatives against T. vaginalis. Methods: Thiazolidines were synthesized via multicomponent reaction (MCR) using one-pot methodology and tested in vitro against the parasite and mammalian cell lines. Results: Seventy percent of the compounds showed more than 80% antiparasitic activity at 100 μM, with compounds 4a, 4b, and 4f exhibiting IC₅₀ ≤ 20 µM. None of the molecules exhibited cytotoxic against Vero CCL-81 and HeLa cells. Evaluation of the structure–activity relationship (SAR) indicates that the substituent at the nitrogen position of the heterocycle may be involved in the antiparasitic effect of these compounds. In silico studies also revealed that the three compounds possess adequate oral bioavailability and do not present mutagenic, tumorigenic or irritating risks. Finally, molecular docking predicted strong interactions of compounds 4a, 4b, and 4f with T. vaginalis enzymes lactate dehydrogenase and purine nucleoside phosphorylase; compound 4f also interacted with methionine Ƴ-lyase. Conclusions: These preliminary results suggest that 1,3-thiazolidin-4-ones are promising scaffolds for developing new trichomonacidal agents. Full article

Background/Objectives: Breast cancer is a prevalent and heterogeneous disease with multiple subtypes, which are defined by characteristics such as molecular biomarkers and metastatic status. This study aimed to profile the metabolic activity of various breast cancer subtypes, both with and without chemotherapy (doxorubicin) application. Methods: Six human breast cell lines were evaluated, two non-tumorigenic controls and four cancerous lines. The cancer lines were clustered as primary-derived, metastasis-derived, triple-negative (TNBC), and strong hormone receptor-positive (ER+/PR+) and analyzed using the Biolog phenotype mammalian microarrays (PM-M1 to PM-M8) to assess metabolic activity via NADH production under a wide array of substrate parameters. Results: Unique metabolic profiles emerged across the subtypes and clusters; the TNBC and metastatic cells demonstrated enhanced utilization of glycolytic and anaerobic substrates consistent with the Warburg effect. The ER+/PR+ cells showed heightened glucose utilization and unique sensitivity to metabolic effectors and doxorubicin. Additionally, significant metabolic differences were observed in nucleoside and amino acid utilization between cancer and control cells, particularly in metastatic and TNBC lines. Conclusions: Our findings reveal the profound metabolic diversity among breast cancer subtypes and highlight distinct substrate dependencies for proliferation. The results additionally provide a framework for developing metabolic biomarkers and targeted therapies for chemotherapy resistance in breast cancer subtypes. Full article

Herpes simplex viruses (HSV-1 and HSV-2) are highly prevalent human pathogens that establish lifelong latency in sensory neurons, posing a persistent challenge to global public health. Their clinical manifestations range from mild, self-limiting orolabial lesions to severe, life-threatening conditions such as disseminated neonatal infections, focal encephalitis, and herpetic stromal keratitis, which can lead to irreversible corneal blindness. Beyond direct pathology, HSV-mediated genital ulcerative disease (GUD) significantly enhances mucosal susceptibility to HIV-1 and other sexually transmitted infections, amplifying co-infection risk and disease burden. Despite decades of clinical reliance on nucleoside analogues such as acyclovir, the therapeutic landscape has stagnated with rising antiviral resistance, toxicity associated with prolonged use, and the complete inability of current drugs to eliminate latency or prevent reactivation continue to undermine effective disease control. These persistent gaps underscore an urgent need for next-generation antivirals that operate through fundamentally new mechanisms. Marine ecosystems, the planet’s most chemically diverse environments, are providing an expanding repertoire of antiviral compounds with significant therapeutic promise. Recent discoveries reveal that marine-derived polysaccharides, sulfated glycans, peptides, alkaloids, and microbial metabolites exhibit remarkably potent and multi-targeted anti-HSV activities, disrupting viral attachment, fusion, replication, and egress, while also reshaping host antiviral immunity. Together, these agents showcase mechanisms and scaffolds entirely distinct from existing therapeutics. This review integrates emerging evidence on structural diversity, mechanistic breadth, and translational promise of marine natural products with anti-HSV activity. Collectively, these advances position marine-derived compounds as powerful, untapped scaffolds capable of reshaping the future of HSV therapeutics. Full article

Phenylketonuria (PKU) is a rare inherited metabolic disorder caused by phenylalanine hydroxylase deficiency, leading to phenylalanine (Phe) accumulation and neurological dysfunction if untreated. While metabolomics holds promise for biomarker discovery in PKU, few studies have examined urinary metabolites using untargeted approaches. This study applied untargeted metabolomics using HPLC-QTOF-MS to analyze urine from 36 adult patients with PKU and 34 healthy controls. Biomarker Analysis was performed with MetaboAnalyst 6.0. A total of 73 significant metabolites (FDR < 0.05; VIP > 1) were identified, with 29 upregulated and 44 downregulated in PKU. A 23% of these metabolites were related to Phe metabolism, while 77% were associated with alterations across more than 10 metabolic pathways, including leucine and tryptophan metabolism, acylcarnitines, vitamins, and diet- or microbiota-derived compounds, among others. Specifically, upregulated metabolites with an AUC > 0.9 included several Phe-derived compounds, the nucleoside 8-hydroxy-7-methylguanine, and indole compounds (1H-indole-3-carboxaldehyde). Conversely, downregulated metabolites with an AUC > 0.9 included N-acetyl(iso)leucine and a heptenoylcarnitine isomer. The Random Forest-based model demonstrated enhanced predictive performance when integrating 10 metabolites, supporting their potential utility as biomarkers for PKU. These findings improve the biological understanding of metabolic disturbances beyond Phe, and may support the development of new therapeutic and dietary strategies. Full article

Background/Objectives: The emergence of drug-resistant HIV-1 strains challenges the long-term efficacy of current antiretroviral therapies. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are critical in HIV-1 treatment; however, the need for new candidates with improved resistance profiles and pharmacokinetics remains. This study aims to design and evaluate novel NNRTIs targeting both wild-type (WT) and mutant-type (MT) HIV-1 reverse transcriptase (RT) using integrated computational strategies. Methods: We conducted a 3D-QSAR study on 33 naphthyl-diarylpyrimidine derivatives using CoMFA and CoMSIA models. We designed thirty-five novel molecules based on contour map insights. We applied ADMET and drug-likeness filters to prioritize ten candidates. Molecular docking was performed on WT (PDB: 3HVT) and MT (PDB: 4PUO) RT structures. The top candidates underwent 100 ns molecular dynamics (MD) simulations. We analyzed structural stability via RMSD, RMSF, and Rg, while we used SASA and MolSA to assess solvent exposure and surface compactness. Results: The CoMFA and CoMSIA models demonstrated robust predictivity (R² = 0.979/0.920, Q² = 0.643/0.546, R²_test = 0.747/0.603). P14 and P43 showed higher binding affinities than nevirapine and favorable ADMET profiles. MD simulations confirmed stable binding in WT-RT and adaptive flexibility in MT-RT. SASA and MolSA analysis revealed favorable conformational compaction. Drug-likeness profiles indicated optimal log P, strong hydrogen bonding, and acceptable bioavailability. Conclusions: P14 and P43 demonstrate strong potential as NNRTI leads, combining binding affinity, structural stability, and favorable pharmacokinetics, supporting further experimental development. Full article

The problem of antibiotic resistance is one of the challenges that science and medicine face in the 21st century. Nucleoside analogs have already proven as antiviral and antitumor agents, and, currently, there are more and more reports on their antibacterial and antifungal activity. The substitution of an oxygen atom by a sulfur one leads to the emergence of unique properties. Here, we report the synthesis of eight new 4-thioanalogs of 5-substituted (5-alkyloxymethyl and 5-alkyltriazolylmethyl) derivatives of 2′-deoxyuridine and uridine, which were active against Mycobacterium tuberculosis and Gram-positive bacteria. The novel sulfur-containing nucleosides were synthesized via activation of the pyrimidine C4 position, followed by condensation with thioacetic acid and deblocking. To increase the solubility, oligoglycol carbonate depot forms were obtained via activation of the 3′-hydroxyl group using N,N’-carbonyldiimidazole and condensation with triethylene glycol. The highest inhibitory activity was demonstrated by 3′-triethylene glycol depot forms of 4-thio-5-undecyl- and 5-dodecyloxymethyl-2′-deoxyuridine (4a,b) against two strains of M. smegmatis. The most promising compounds were 5-[4-decyl-(1,2,3-triazol-1-yl)methyl]-4-thio-2′-deoxy- and ribouridine (3c,g) and 5-undecyloxymethyl 4-thiouridine (3e) active toward clinical M. intracellulare isolates. Overall, novel sulfur-containing nucleoside analogs were low toxic, demonstrated better inhibitory activity compared to their C4-oxo ones, and, thus, are promising compounds for the development of new antibacterial agents. Full article

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important DNA repair enzyme and its functioning is considered as one of the possible reasons for tumor resistance to topoisomerase 1 (TOP1) poisons such as topotecan. Thus, TDP1 inhibitors in combination with topotecan may improve the effectiveness of anticancer therapy. TDP1 acts somehow in a phospholipase manner, depleting the phosphodiester bond between lipophilic tyrosine residue and 3′ end of DNA; therefore, lipophilic molecules bearing aromatic substituents can interact with TDP1 and even possess high inhibitory activity, which is evidenced by data from the literature. Previously, we identified lipophilic nucleoside derivative (compound 6d, IC₅₀ = 0.82 µM) as an effective inhibitor of the purified enzyme TDP1 that enhances the cytotoxic, DNA-damaging, and antitumor effects of topotecan. However, the role of TDP1 inhibition in this synergistic effect remained not fully understood. In the present study, we have tested the hypothesis of a TDP1-dependent mechanism of action for compound 6d, showing that it sensitizes wild-type A549 lung cancer cells, but not TDP1 knockout cells, to the cytotoxic effects of topotecan. The sensitizing effect was absent in non-cancerous HEK293A cells regardless of TDP1 status. Additionally, we analyzed the effect of compound 6d and topotecan on the expression level of TOP1 and TDP1 to determine whether the observed synergy was due to direct TDP1 inhibition and/or changes in regulation of these enzymes. The data obtained shows that compound 6d did not affect TDP1 gene expression level in HEK293A and A549 WT cells. Thus, compound 6d most probably does not suppress the transcription or mRNA stability of TDP1, and the synergistic action of 6d with topotecan is related to TDP1 inhibtion. Full article

The gut microbiome possesses a diverse range of biological properties that play a role in maintaining host health and preventing disease. Gut microbial metabolites, including short-chain fatty acids, natural purine nucleosides, ellagic acid derivatives, and tryptophan metabolites, have been observed to have complex and multifaceted roles in the gut and in wider body systems in the management of disease, including cancer. Triple-negative breast cancer is the most aggressive subtype of breast cancer, with restricted treatment options and poor prognoses. Recently, preclinical research has investigated the antiproliferative potential of gut microbial metabolites against this type of breast cancer with promising results. However, little is understood about the mechanisms of action and molecular pathways driving this antiproliferative potential. Understanding the complex mechanisms of action of gut microbial metabolites on triple-negative breast cancer will be instrumental in the investigation of the combined administration with standard chemotherapeutic drugs. To date, there is a paucity of research studies investigating the potential synergistic interactions between gut microbial metabolites and standard chemotherapeutic drugs. The identification of synergistic potential between these compounds may provide alternate and more effective therapeutic options in the treatment and management of triple-negative breast cancer. Further investigation into the mechanistic action of gut microbial metabolites against this breast cancer subtype may support the administration of more cost-effective treatment options for breast cancer, with an aim to reduce side effects associated with standard treatments. Additionally, future research will aim to identify more potent metabolite–drug combinations in the mitigation of triple-negative breast cancer progression and metastasis. Full article

Hydroethanolic Cucurbita pepo seed extracts are traditionally used for alleviating lower urinary tract symptoms (LUTS), yet their mechanisms remain unclear. Adenosine, a purine nucleoside involved in neuromodulation and smooth muscle relaxation, was recently identified in C. pepo seeds. Since A₁ adenosine receptors (A₁AR) suppress parasympathetic bladder overactivity by inhibiting acetylcholine (ACh) release, we investigated to which extent purines from pumpkin seed extracts contribute to A₁AR activation. Complementary antioxidant capacity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Three hydrophilic seed extracts containing different adenosine levels (0.60–1.18 mg/g dw) were evaluated for agonist activity using a cAMP inhibition assay. The most active extract showed an EC₅₀ of 40.22 µg/mL. Selective removal of adenosine shifted the dose–response curve rightward, while further elimination of an adenosine derivative increased the EC₅₀ to 212.10 µg/mL, confirming adenosine as the principal active compound. Guanosine and inosine did not exhibit A₁AR agonist or allosteric effects. All samples exhibited measurable but weak antioxidant activity (IC₅₀ = 1.02–4.19 mg/mL), consistent with their low total phenolic content. These findings underscore the importance of accounting for naturally occurring agonists in plant extracts to avoid overestimating receptor-mediated effects in vitro which are not translatable in vivo. Full article

Background and Objectives: Endometrial adenocarcinoma is among the most prevalent malignancies of the female reproductive system, and therapeutic options remain limited, particularly in advanced stages. In recent years, natural agents, especially flavonoids, have gained considerable interest for their capacity to enhance the effectiveness of chemotherapeutic drugs and modulate tumor-related molecular mechanisms. Hesperidin, a citrus-derived flavonoid, is recognized for its antioxidant and anti-inflammatory effects, while Gemcitabine, a nucleoside analog, is widely used in cancer treatment. Investigating their combined effects on endometrial carcinoma cells could yield novel insights into multimodal therapeutic development. This current study aimed to assess the impact of Hesperidin (Hes) and Gemcitabine (Gem) on ISHIKAWA cells, a human endometrial adenocarcinoma model, with particular attention to pathways associated with hypoxia, angiogenesis, apoptosis, and oxidative stress. Materials and Methods: ISHIKAWA cells were treated with varying concentrations of Hes (50–200 µM) and Gem (10–50 nM), either individually or together, for 24 and 48 h. Cell viability was determined using the MTT assay, while apoptosis was measured by Caspase-3/7 activity and NucBlue nuclear staining. Intracellular reactive oxygen species (ROS) generation was quantified via DCFH-DA fluorescence. Expression levels of HIF-1α, VEGF, Bax, Bcl-2, and Caspase-3 were examined by RT-qPCR. Synergistic interactions were analyzed with the Chou–Talalay combination index. Biological enrichment was further explored using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results: Both Hes and Gem significantly decreased ISHIKAWA cell viability in a concentration- and time-dependent manner (p < 0.001). The combined treatment induced stronger apoptotic effects, as reflected by increased Caspase-3/7 activity and nuclear morphological changes. RT-qPCR demonstrated upregulation of Bax and Caspase-3, together with downregulation of Bcl-2, HIF-1α, and VEGF. While Hes reduced intracellular ROS, Gem elevated it; their combination produced a balanced oxidative response. All dose combinations displayed strong synergism (CI < 1). GO and KEGG enrichment confirmed the involvement of apoptosis-, angiogenesis-, and hypoxia-related pathways. Conclusions: Co-treatment with Hes and Gem exhibits synergistic anticancer activity in endometrial cancer cells by promoting apoptosis, suppressing angiogenesis- and hypoxia-related gene expression, and modulating oxidative stress. This combined therapeutic approach highlights its potential as a promising adjuvant option, warranting further evaluation in in vivo and translational studies. Full article

Background: Type 2 diabetes (T2D), the most common form of diabetes, is associated with a significantly elevated risk of cardiovascular and cerebrovascular complications. However, circulating metabolic signatures that reliably predict the transition to insulin resistance, and are potentially linked to increased vascular risk, remain incompletely characterized. Rodent models, particularly those induced by a high-fat diet (HFD) combined with low-dose streptozotocin (STZ), are widely used to study the progression of T2D. However, the systemic metabolic shifts associated with this model, especially at the plasma level, are poorly defined. Methods: In this study, we performed untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomic profiling on plasma samples from control, HFD-only (obese, insulin-sensitive), and HFD + STZ (obese, insulin-resistant) C57BL/6 mice. Results: In the HFD + STZ cohort, plasma profiles showed a global shift toward lipid classes; depletion of aromatic and branched-chain amino acids (BCAAs); accumulation of phenylalanine-derived co-metabolites, consistent with gut–liver axis dysregulation; elevations in glucose, fructose-6-phosphate, and nucleoside catabolites, indicating impaired glucose handling and heightened nucleotide turnover; increased free fatty acids, reflecting membrane remodeling and lipotoxic stress; and higher cAMP, thyroxine, hydrocortisone, and uric acid, consistent with endocrine and redox imbalance. By contrast, HFD-only mice exhibited elevations in aromatic amino acids and BCAAs relative to controls, a pattern compatible with early obesity-associated adaptation while insulin signaling remained partially preserved. KEGG analysis revealed disturbances in carbohydrate metabolism, amino acid degradation, nucleotide turnover, and hormone-related pathways, and HMDB mapping linked these changes to T2D, obesity, heart failure, and renal dysfunction. Conclusion: Collectively, these findings delineate insulin resistance-specific plasma signatures of metabolic inflexibility and inflammatory stress in the HFD + STZ model, distinguishing it from HFD alone and supporting its utility for mechanistic studies and biomarker discovery. Importantly, this plasma metabolomics study shows that insulin-sensitive and insulin-resistant states exhibit distinct variation in circulating metabolites and cardiovascular risk factors, underscoring the translational value of plasma profiling. Full article

Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-α and nucleoside/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently. Full article