Using multi-spectroscopy and molecular docking techniques, the binding mechanism between two azo dyes and the calf thymus (Ct-DNA) was investigated under physiological settings. After the addition of Ct-DNA, each azo dye exhibited a hypochromic effect and slightly increased the wavelength of the maximum absorption. This suggested that the probes and Ct-DNA interacted via a groove binding mode, which was corroborated by the molecular docking data. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, were determined by calculating the binding constants from the maximum absorption spectra of both azo dyes at different Ct-DNA concentrations at different temperatures. Moreover, fluorescence resonance energy transfer indicates that the interval between the donor (EB-Ct-DNA) and acceptor (azo dye) is suitable for energy transfer. Hydrogen bonds and π electrons on the azo dye's benzene ring are essential for binding the dye to Ct-DNA, according to molecular modeling research. These probes bind to the minor groove of Ct-DNA, and the molecular docking results are in good agreement with the spectroscopic findings.
It was previously demonstrated that (E)-5',6'-didehydro-6'-deoxy-6'-fluorohomoadenosine (EDDFHA) undergoes addition of water across the isolated C5'-C6' double bond by S-adenosyl-L-homocysteine (AdoHcy) hydrolase without prior oxidation at C3' (J. Biol. Chem.1993, 268, 17030). Addition of water at the 5' position results in the formation of 6'-deoxy-6'-fluoro-5'-hydroxyhomoadenosine (DFHHA). We now describe preparation of EDDFHA, its Z-isomer and their 6'-[2H]-labeled analogues as well independent syntheses of DFHHA and 6'-deuterio-DFHHA. These compounds allow the assignment of absolute configuration at the 5' carbon atom of DFHHA as well as a determination of the overall stereochemistry of enzymatic addition of water at the 5' carbon atom and protonation at the 6' carbon atom.
Oligonucleotide therapeutics represent a rapidly advancing class of medicines that has attracted substantial attention in recent years. Phosphoramidites with chemical modifications are essential building blocks for their chemical synthesis. Therefore, a deep understanding about the stability of phosphoramidites is crucial for the manufacturing of oligonucleotide therapeutics. Especially, it is known that the DNA phosphoramidite with isobutyryl protected-guanine nucleobase (dG[iBu]) is unstable in anhydrous solution. In this study, the solution stabilities of dG[iBu] phosphoramidites with sugar modification were evaluated. The sugar-modified phosphoramidites in acetonitrile were found to be more stable than DNA phosphoramidites, probably due to steric hindrance of the modifications at their 2'-position. Moreover, N,N-dimethylformamide and dichloromethane were identified as alternative solvents that could improve stability without affecting the reactivity of phosphoramidites in oligonucleotide synthesis.
Triple-negative breast cancer (TNBC) is the most invasive subtype of breast cancer (BC) with high recurrence, high mortality, poor prognosis, and a lack of effective targeted therapies. This study aimed to investigate the prognostic value of lncRNA Alu-mediated p21 transcriptional regulator (APTR) in TNBC. APTR/miR-298 levels in TNBC tissues and cell lines were detected by RT-qPCR. The correlation between APTR and the prognosis of TNBC patients was analyzed by Kaplan-Meier survival analysis. The direct binding relationship between APTR and miR-298 was verified with a luciferase reporter assay. CCK-8 and Transwell assays were conducted to elucidate the role of APTR knockdown and miR-298 co-knockdown in regulating the viability and migration of TNBC cells. APTR was upregulated in TNBC tissues and cell lines, while miR-298 was downregulated. High APTR expression was closed associated with poor prognosis of TNBC patients. Mechanistically, APTR directly binds to and negatively regulates miR-298. Functional experiments showed that knockdown of APTR significantly inhibited the viability and migration of TNBC cells, while miR-298 inhibition partially abrogated these tumor-suppressive effects. APTR is upregulated in TNBC and is closely related to poor prognosis. APTR exerts an oncogenic role in TNBC by negatively regulating miR-298, suggesting that APTR serves as a promising novel prognostic biomarker for TNBC.
This work examined the kinetics for the B→Z-DNA transition of tandem d(CG) repeat sequences induced by NaCl, KCl, MgCl2, and spermine. While d(CG)3 undergoes an incomplete transition, d(CG)6, d(CG)9, and d(CG)18 transition fully in appropriate concentrations of salts or spermine. The transition proceeds more slowly with increasing DNA lengths. Furthermore, d(CG)18 displays complex behavior in the presence of MgCl2. While d(CG)18 fully transitions to Z-DNA in the presence of 2.5 M MgCl2, the process is complicated, likely by condensation. Kinetics of the B-/Z-DNA transition is an important aspect of Z-DNA's roles in biological systems. The knowledge of how this transition is affected by sequence lengths and salts helps define the DNA's conformational plasticity in response to environmental shifts in cells.
A series of nucleoterpenoids in which uridine is attached to diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) by means of an alkyl or 1,2,3-triazolylalkyl linker has been synthesized. Screening of their in vitro cytotoxicity against 7 cancer cell lines revealed three lead compounds. This is nucleoterpenoid 13c in which the uridine moiety is attached by the octyl linker to the amide group of isosteviol. Compound 13c caused the death of MCF-7 and PANC-1 cancer cells at an IC50 concentration of 11 µM. Nucleoterpenoid 15b, in which the uridine moiety with protected hydroxyl groups is attached by the 1,2,3-triazolylbutyl linker to the C-16(S) position of isosteviol, caused the death of cancer cells M-HeLa, MCF-7, PANC-1, PC-3, A 549, and HuTu 80 at IC50 values in the range 10.4-16.8 µM. Its derivative, nucleoterpenoid 15d with free hydroxyl groups, caused the death of cancer cells MCF-7, PANC-1, PC-3, and A 549 at IC50 values in the range 11.7-19.5 µM.
Associations of the MICA (rs2596542), GPC3 (rs2267531), PNPLA3 (rs738409), and TM6SF2 (rs58542926) gene variants with the susceptibility to developing Hepatocellular Carcinoma (HCC) were studied. Blood samples from 600 subjects, including 300 HCC patients and 300 age- and gender-matched controls, were collected from different hospitals in Punjab, Pakistan. Genomic DNA was extracted from blood, and PCR was performed. The results showed that the mutant (TT) of the rs2596542 of the MICA gene is significantly associated with a decreased risk of HCC, while the heterozygote (CT) of the same polymorphism shows a significantly high association with increased HCC risk of 2-fold. In the case of the rs2267531 polymorphism of the GPC3 gene, the mutant (GG) is significantly associated with decreased risk of HCC. In contrast, the heterozygous genotype (AG) of this polymorphism exhibited a highly significant association with a 7-fold higher risk of liver cancer. In the rs738409 variants of gene PNPLA3, heterozygous (AG) has a highly significant association with hepatocellular carcinoma, with increased risk of hepatocellular carcinoma by 2-fold. In rs58542926, a homozygous mutant (GG) also shows a substantial correlation with HCC, with increased risk of HCC up to 2-fold. In contrast, the heterozygous (AG) genotype of the rs58542926 SNP shows a highly significant relationship with decreased risk of HCC.
Long non-coding RNA (lncRNA) EGOT is dysregulated in multiple malignancies and participates in tumor progression. Its role and mechanism in lung adenocarcinoma (LUAD) were unclear. The present study set out to investigate the expression pattern of EGOT in LUAD, clarify its clinical significance, and explore the molecular mechanism regulating LUAD cell malignant phenotypes. Tumor tissues and their corresponding adjacent normal tissues were obtained from a cohort of 130 LUAD patients. EGOT expression was detected by RT-qPCR. LUAD cell models with EGOT overexpression or knockdown were established. CCK-8, Transwell, and flow cytometry assays were employed to assess LUAD cell phenotypes. Bioinformatics prediction, pull-down, and dual-luciferase reporter assay were performed to authenticate the regulatory axis. EGOT was downregulated in LUAD tissues and cells (A549 and PC9), and low EGOT expression was closely associated with poor prognosis and adverse clinical features (advanced TNM stage, increased tumor size, and lymph node metastasis). EGOT impaired the proliferative, migratory and invasive capacities of LUAD cells, and facilitated their apoptotic process. EGOT targeted miR-1323 to regulate QKI expression, forming the EGOT/miR-1323/QKI axis in A549 and PC9 cells. MiR-1323 reversed EGOT's effect in LUAD cells, which was abolished by QKI overexpression. EGOT may act as a tumor suppressor in LUAD via the miR-1323/QKI axis, providing a potential prognostic biomarker for LUAD.
Pancreatic ductal adenocarcinoma (PDAC) is intrinsically drug-resistant. Combinations of 5-fluorouracil (5-FU) with other cytostatics in FOLFIRINOX and gemcitabine (Gem) with nab-paclitaxel are first-line treatments. Colorectal cancer (CRC) is treated with FOLFOX or FOLFIRI, but in advanced disease patients become resistant for whom Gem has been considered. We investigated whether combining the key drugs, 5-FU and Gem, would be advantageous with different treatment schedules. We used CRC (human WiDr cells and Colon 26-A, a syngeneic mouse tumor) and a murine PDAC (PANC02 cells and tumors) as well-defined model systems. In WiDr at 3-day simultaneous exposure, the 5-FU and Gem combination was antagonistic, while in PANC02 cells it was additive at simultaneous addition, but synergistic when Gem was added 6 hr after 5-FU. The antagonism might relate to the decrease in FdUMP concentration (about 50%) caused by Gem. Based on these data, we initiated an in vivo study with Gem administered after 5-FU. Since 5-FU is more effective as a prolonged infusion, we used 5-FU pellets, implanted subcutaneously, to deliver 5-FU for 5 days. Gem was given at q3dx4. The 2.5 mg 5-FU infusion with 75 mg/kg Gem was most optimal with tolerable weight loss (<10%). The tumor size of treated vs control tumors (T/C) at this schedule for CRC was 0.49 for 5-FU alone, 0.21 for Gem alone, and 0.16 for the combination. For PDAC, these values were 0.77, 0.74, and 0.49, respectively. In conclusion, unfortunately, the efficacy of the combination of 5-FU with gemcitabine was moderate, both in vitro and in vivo.
Metabolic syndrome (MetS) is a composite cardiometabolic condition defined by visceral obesity, dyslipidemia, hypertension, and insulin resistance, and it promotes the worldwide prevalence of type 2 diabetes and atherosclerotic cardiovascular disease. Nucleic-acid aptamers have developed as programmable recognition ligands that integrate antibody-like specificity with chemical synthesis, modular functionalization, low batch variability, and usually a low immunogenicity. Their translational significance in MetS is not solely in target binding, but in the capacity to establish disease-relevant affinity, stability, and tissue selectivity by advanced SELEX, counter-selection, chemical expansion, truncation, and post-SELEX maturation. Aptamers can function as pathway modulators by inhibiting ligand-receptor interactions, retaining biased receptor conformations, or concentrating therapeutic agents in metabolically compromised tissues. This provides them particularly appealing for targets characterized by receptor recycling, cell-state identification, or inflammatory interactions, rather than by a singular static biomarker. They can serve as sensitive biosensing components in aptasensors for glucose, insulin, glycated proteins, lipids, and inflammatory markers, offering more detailed metabolic phenotyping than single-analyte assays. Recent studies on insulin receptor, PCSK9, adipocyte-, and hepatocyte-targeting aptamers illustrate how aptamers may support both disease-modifying therapy and point-of-care diagnostics in obesity, insulin resistance, MASLD, and dyslipidemia. Nevertheless, matrix-aware validation, serum stability, pharmacokinetic optimization, and proof of superiority over well-known biologics and clinical tests are still necessary for effective translation. Aptamer engineering offers a versatile and mechanistically adaptable framework for precision diagnostics along with targeted intervention in Metabolic syndrome. If these challenges be successfully overcome, aptamers may transition from sophisticated binders to clinically applicable metabolic tools.
Vitamin B12 (B12) deficiency may cause severe clinical complications, partly attributable to the "methylfolate trap". When B12-dependent methionine synthase activity is impaired, 5-methyltetrahydrofolate (5-MTHF) becomes trapped because it cannot be used in the methionine synthase reaction or reconverted to 5,10-methylenetetrahydrofolate. This reduces the availability of folate cofactors, including tetrahydrofolate (THF), 5,10-methylene-THF, and 10-formyl-THF required for histidine catabolism, pyrimidine metabolism, and de novo purine synthesis (DNPS). We investigated whether this mechanism is reflected in urinary DNPS intermediates. Using LC-MS/MS, we quantified the histidine catabolite formiminoglutamate (FIGLU) and urinary DNPS intermediates in 28 patients with confirmed B12 deficiency and age-stratified controls. Serum methylmalonic acid (MMA) served as a proxy for B12 deficiency severity. Compared with controls, urinary FIGLU and DNPS intermediates-glycinamide ribonucleoside (GAr) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr)-were significantly increased across all age groups (p < 0.05), while succinyl-5-aminoimidazole-4-carboxamide ribonucleoside (SAICAr) was also increased, except in adults. These changes were more evident in severe B12 deficiency-defined as MMA ≥ 10× the age-specific upper limit (UL): THF depletion was reflected by marked FIGLU elevation (up to several dozen multiples of UL), while reduced folate cofactor availability was demonstrated by increased GAr, SAICAr, and AICAr (up to 3.4-, 2.7-, and 5.0-fold UL, respectively). In linear correlation analyses, urinary FIGLU correlated with serum MMA, and GAr, SAICAr, and AICAr correlated with FIGLU, a marker of THF deficiency (p < 0.05). These findings are consistent with methylfolate trapping, linking B12 deficiency to reduced availability of folate cofactors for histidine catabolism and DNPS.
Fluorocyclopentenylcytosine (RX-3117) is a nucleoside analog developed to bypass gemcitabine resistance. We developed several fluorocyclopentenylcytosine- and gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines, which showed decreased accumulation of the active triphosphate. The expression of the human equilibrative nucleoside transporter and the activity of uridine cytidine kinase 2 were not altered. The cells showed cross-resistance to some cytidine analogs (e.g. ethynylcytidine and cyclopentenyl-cytosine) but not to other DNA damaging drugs, such as cisplatin, and drugs inhibiting the proteasome, a protease or cell cycle regulators. In most resistant lines, the expression of MutT Homolog-1/8-oxo-dGTP diphosphatase (MTH1) and deoxycytidine triphosphatase 1 (DCTPP1) was increased, suggesting that these enzymes degrade the triphosphates. To reverse resistance, we combined fluorocyclopentenylcytosine with inhibitors of MTH1 (TH588) and DCTPP1 (TH1217 and triptolide). Drug sensitivity was tested in resistant NSCLC variants using the SRB assay with co-treatment of fluorocyclopentenylcytosine and IC25 concentrations of inhibitors. Despite the increased expression of MTH1 and DCTPP1, treatment with TH588, TH1217, or triptolide did not re-sensitize the resistant lines, except for cells resistant to gemcitabine and to both gemcitabine and fluorocyclopentenylcytosine, which showed increased sensitivity to fluorocyclopentenylcytosine combined with the inhibitors. In conclusion, while fluorocyclopentenylcytosine resistance involves decreased triphosphate accumulation and increased expression of DNA repair enzymes, only gemcitabine- and double-resistant cells became more sensitive to enzyme inhibition, suggesting other mechanisms contribute to resistance.
Circular RNAs (circRNAs) have arisen as a new area of interest among non-coding RNAs in cancer. Increased stability, abundance in human cells, tissue specificity, and regulatory functions in various diseases including cancer indicate that circRNAs may serve as important biomarkers. Oral squamous cell carcinoma (OSCC), the leading form of oral cancer, exhibits decreased survival, high recurrence, and mortality rates, and has been a significant public health concern for a long time. The molecular mechanisms involved in OSCC are not well understood, and there is a significant lack of accurate biomarkers for its timely identification, and prognosis, along with treatment targets. Existing literature suggests that circRNAs are implicated in various aspects in OSCC carcinogenesis and reveal their potential role in the detection and management of disease. The interaction between circRNAs, and miRNAs induces modulation of multiple signaling pathways, and assists in distinct cancer characteristics like generating own growth signals, angiogenesis, infinite proliferation, tissue invasion, and metastasis. This review summarizes protumoal, and antitumoral properties of circRNAs, their role as prospective biomarkers, and therapy targets along with their involvement in signaling pathways impacting OSCC tumorigenesis, and progression.
Neurodegenerative diseases are associated with progressive neural malfunction, which is driven by common molecular pathologies that encompass protein aggregations, mitochondrial dysfunction, aberrant RNA metabolism and impaired intracellular clearance. Conventional treatments are largely symptomatic with no treatment of the underlying pathology. Gene therapies, RNA-based therapeutic platforms and CRISPR-based genome-editing technologies provide more targeted methods to regulate the pathological pathways and restore neuronal homeostasis. Nevertheless, these interventions can have transient, reversible or long-term effects instead of a consistent irreversible effect depending on the platform being used. Engineered viral vectors, particularly adeno-associated viruses, enable cell-type-specific and circuit-resolved delivery within the central nervous system. Although constrained by a limited packaging capacity (∼4.7 kb), innovations such as dual-vector systems and capsid engineering are expanding their functional utility. RNA therapeutics, such as antisense oligonucleotides, siRNA/miRNA and synthetic mRNA, provide reversible gene expression regulation, whereas CRISPR can be used to disrupt, correct or regulate the expression of specific genes. Together, these platforms constitute a multifaceted and evolving toolkit for neuroprotection, with the potential to modify disease progression in neurodegenerative disorders. However, most approaches remain at preclinical or early clinical stages, and further validation is required to establish long-term efficacy and safety.
To achieve efficient cholinesterase inhibitory activity of metal(II) complexes of Cu(II), Ni(II), Co(II), and Zn(II) with 2,2'-bipyridyl framework [M-L] (L = 2,2'-bipyridyl derivative containing an aromatic center and an e--withdrawing -NO2 group) was developed. The structural characteristics were identified through spectroscopic and analytical studies. The antibacterial activity of the produced ligand and metal(II) complexes against bacteria and fungi was evaluated. The synthesized metal(II) complexes ability to fragment DNA has been studied on pUC 18 DNA using agarose gel electrophoresis. The copper(II) complex (Kb=4.11 × 105 M-1) is stronger binding affinity for DNA than ethidium bromide (EB) (Kb=3.3 × 105 M-1) and metal(II) complexes. The chemically produced 2,2'-bipyridyl derivative had the strongest inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 values that were less than the standard compounds (0.34 and 3.42 µM, respectively). Our research results could aid in the creation of novel drug molecules, especially for the treatment of neurological conditions like Alzheimer's disease and neurological disorders occurring through diabetes.
Gram-positive pathogen Staphylococcus aureus is associated with human mortality and morbidity worldwide. Mutants of this pathogen is responsible for life threatening community-associated meticillin-resistant infections. Further, the evolution of multidrug-resistant Staphylococcus aureus strains emphasizes the urgent need to develop novel antibacterial agents. ATP-dependent D-alanyl:D-alanine ligase is essential to produce peptidoglycan of bacterial cells, and its inhibition can lead to bacterial cell death, making it a valuable antibacterial target. D-alanyl-D-alanine ligase is recognized as a validated target for the design and development of novel antibacterial agents to overcome resistance problems. In the past several approaches have been used to develop D-alanyl-D-alanine ligase inhibitors with potent antibacterial activity. Some of these inhibitors, including N-acyl-substituted sulfamides, 1-(2-hydroxybenzoyl)-thiosemicarbazide, benzoylthiosemicarbazide, benzoxazoles and diazenedicarboxamides exhibited significant activity against Staphylococcus aureus. The present findings clearly demonstrate that inhibiting D-alanyl:D-alanine ligase is a viable strategy for the discovery of new antibacterial therapies against Staphylococcus aureus.
A series of novel pyrimidine-thiadiazole conjugates incorporating carbohydrate-based 1,3,4-thiadiazole thioglycosides were synthesized and analyzed using (IR), (1H-NMR) and (13C-NMR) spectroscopy. The synthesized compounds were evaluated for their cytotoxic activity against human liver (HepG2), breast (MCF-7), and lung (A549) cancer cell lines. Compound 10 showed the strongest activity with IC50 values of 10.73 ± 2.04 mM (HepG2), 9.10 ± 1.97 mM (MCF-7), and 9.25 ± 2.94 mM (A549). while 6 and 7 demonstrated moderated activity. In contrast, the reference drug doxorubicin exhibited higher potency, with IC50 values of 3.71 ± 1.12 mM (HepG2), 2.68 ± 0.11 mM (MCF-7), and 3.22 ± 0.22 mM (A549). Molecular docking studies revealed compound 10 had favorable binding profile with an S-score of -6.33 kcal/mol and RMSD of 3.10 Å, forming key interactions including hydrogen bonding with GLU 142, H-acceptor interaction with GLY 104, and π-H interaction with HIS 141. Compound 5 also exhibited strong binding interactions (S-score = -5.87 kcal/mol) with multiple metal and ionic interactions, whereas compounds 6, 7, and 11 displayed comparatively weaker binding affinities. These results indicate that compound 10 is the most promising candidate in this series, with both enhanced cytotoxic activity and favorable docking interactions, suggesting that pyrimidine-thiadiazole hybrids represent a valuable scaffold for further anticancer drug development.
The pathogenesis of endometriosis (EM) remains intricate and multifactorial. This study aimed to investigate the biological functions of microRNA-769-5p (miR-769-5p) in an in vitro model of EM. Serum was obtained from 120 EM patients and 100 healthy controls. miRNA and mRNA expression were quantified by RT-qPCR. Functional analyses of miR-769-5p were performed in vitrousing cell counting kit-8 (CCK-8) cell proliferation assays, Transwell assays, and dual-luciferase assays in an endometriosis-derived stromal cell line (hEM15A). miR-769-5p was markedly down-regulated in EM sera and correlated inversely with disease severity (p < 0.001). Moreover, miR-769-5p exhibited high diagnostic accuracy for EM (AUC = 0.9166, p < 0.001). In the hEM15A cell model, overexpression of miR-769-5p effectively suppressed the proliferation, migration, and invasion capabilities of EM-derived stromal cells (p < 0.001). miR-769-5p targeted follistatin (FST) and negatively regulated its expression in vitro (p < 0.001). In contrast, FST overexpression could partially reverse the inhibitory effects of miR-769-5p-mimic on these EM-derived cells. Our findings indicate that miR-769-5p is downregulated in EM serum. Furthermore, in vitro experiments suggest that it can target FST to inhibit proliferation, migration, and invasion of ectopic endometrial stromal cells.
The incidence of cervical cancer (CC) is extremely high, yet current diagnostic biomarkers have not achieved satisfactory results. MicroRNAs can regulate vital processes in tumors, including proliferation and apoptosis. The regulatory role of miR-1247-5p in CC remains unknown. This study aims to investigate the diagnostic value of miR-1247-5p in CC. This study included 156 patients with CC and 130 healthy volunteers. Real-time quantitative PCR was used to detect miR-1247-5p and Dishevelled 1 (DVL1) levels. Cell proliferation was assessed using the CCK-8 assay. Levels of Fe2+, lipid reactive oxygen species, malondialdehyde, and glutathione were measured using commercial kits. Diagnostic value of each biomarker was evaluated via receiver operating characteristic curve analysis. miR-1247-5p is significantly downregulated in CC. Low miR-1247-5p constitutes one of the risk factors for CC development. miR-1247-5p mimic inhibits proliferation and promotes ferroptosis. DVL1 is a target of miR-1247-5p. It is upregulated in both serum from CC patients and CC cells. oe-DVL1 partially reverses the suppression of CC malignant behavior by miR-1247-5p mimic. Therefore, we conclude that miR-1247-5p suppresses the malignant behavior of CC by targeting DVL1 and may serve as a potential clinical diagnostic biomarker.
