Search Results (151)

Background/Objectives: Ewing sarcoma (ES), a highly aggressive bone and soft tissue cancer occurring in children and young adults, is defined by the ETS fusion oncoprotein EWS::FLI1. Although event-free survival rates remain high in ES patients with localized disease, those with metastatic or relapsed disease face poor long-term survival odds. Topoisomerase 1 (TOP1) inhibitors are commonly used therapeutics in ES relapse regimens. Methods: In this work, we used a genome-wide CRISPR knockout library screen to identify the deletion of the TOP1 gene as a mechanism for resistance to topoisomerase 1 inhibitors. Using isogenic cell line models, we performed a high-throughput small-molecule screen to discover a small molecule, GNF-7, which had an IC50 that was 10-fold lower in TOP1-deficient cells when compared to the wild-type cells. Results: The characterization of GNF-7 demonstrated the molecule was highly active in the inhibition of CSK, p38α, EphA2, Lyn, and ZAK and specifically downregulated genes induced by the EWS::FLI1 fusion oncoprotein. Conclusions: Together, these results suggest that GNF-7 or small molecules with a similar kinase profile could be effective treatments for ES patients in combination with TOP1 inhibitors or for those patients who have developed resistance to TOP1 inhibitors. 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

Aromathecin compounds—which contain the same indolizine core structure as camptothecin-like compounds—are expected to show anticancer activity. Among them, 22-hydroxyacuminatine—which has a substituent on the E-ring of the pentacyclic scaffold—exhibits topoisomerase 1 inhibitory activity; therefore, the development of efficient methods for its synthesis has been actively pursued. Herein, we report a versatile synthetic methodology for introducing various substituents on the E-ring, leading to the total synthesis of 22-hydroxyacuminatine as a model compound of the aromathecin family. The synthesis comprises the following key steps: the synthesis of an isoquinoline N-oxide via the thermal cyclization of 2-alkynylbenzaldehyde oxime, the subsequent Reissert–Henze-type reaction to yield an isoquinolone, and the construction of the indolizine moiety (CD-ring) through C–N bond formation via the Mitsunobu reaction. Consequently, a pentacyclic benz[6,7]indolizino[1,2-b]quinolin-11(13H)-one framework is obtained. Using this methodology, the total synthesis of the natural products norketoyobyrine and naucleficine and an intermediate of the latter, which are indoloquinolizidine-type alkaloids, was achieved, and their antiproliferative activity against HCT-116 human colon cancer cells and HepG2 human liver cancer cells was assessed. Naucleficine and its intermediate exhibited moderate antiproliferative activity against HCT-116 cells, with IC₅₀ values of 55.58 and 41.40 μM, respectively. Full article

In this study, we examined the activation of the ATG8, HSP12, KGD1, and POT1 genes in response to decreased glucose levels in the culture medium. Our results show that in top1Δ strains, gene activation is further enhanced compared to WT strains under low glucose conditions, indicating that Top1p represses these genes. This repression occurs independently of its catalytic function. We investigated Rpd3p as an interacting factor of Top1p and found that in rpd3Δ mutants, gene expression under low glucose conditions is even higher than in top1Δ strains, suggesting that Rpd3p also acts as a negative regulator. ChIP analysis revealed that while Top1p levels in regulatory regions remain constant, Rpd3 recruitment increases on promoters after glucose reduction in WT strains but significantly decreases in top1Δ strains. Overall, our findings suggest that Rpd3p is recruited by Top1p to regulate gene expression at controlled physiological levels, highlighting the role of Top1p in transcriptional regulation, controlling helical stress, and interacting with key regulatory factors in response to environmental changes. Full article

Lactation traits are critical economic attributes in domestic animals. This study investigates genetic markers and functional genes associated with lactation traits in Xinjiang donkeys. We analyzed 112 Xinjiang donkeys using 10× whole genome re-sequencing to obtain genome-wide single nucleotide polymorphisms (SNPs). Genome-wide association analyses were conducted using PLINK 2.0 and GEMMA 0.98.5 software, employing mixed linear models to assess several lactation traits: average monthly milk yield (AY), fat percentage (FP), protein percentage (PP), and lactose percentage (LP). A total of 236 SNPs were significantly associated with one or more milk production traits (p < 0.000001). While the two-software identified distinct SNP associations, they consistently detected the same 11, 95, 5, and 103 SNPs for AY, FP, PP, and LP, respectively. Several of these SNPs are located within potential candidate genes, including glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1), FLII actin remodeling protein (FLII), mitochondrial topoisomerase 1 (TOP1MT), thirty-eight-negative kinase 1 (TNK1), polo like kinase 1 (PLK1), notch homolog 1 (NOTCH1), developmentally regulated GTP-binding protein 2 (DRG2), mitochondrial elongation factor 2 (MIEF2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2), and dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). Additionally, we validated the polymorphism of 16 SNPs (10 genes) using Kompetitive Allele Specific PCR, revealing that TOP1MT_g.9133371T > C, GPIHBP1_g.38365122C > T, DRG2_g.4912631C > A, FLII_g.5046888C > T, and PLK1_g.23585377T > C were significantly correlated with average daily milk yield and total milk yield in the studied donkeys. This study represents the first genome-wide association analysis of markers and milk components in Xinjiang donkeys, offering valuable insights into the genetic mechanisms underlying milk production traits. Further research with larger sample sizes is essential to confirm these findings and identify potential causal genetic variants. Full article

Background/Objectives: TDP-43 mutation-driven Amyotrophic Lateral Sclerosis (ALS) motor neuron disease is one of the most prominent forms (approximately 97%) in cases of sporadic ALS. Dysfunctional autophagy and lysosomal function are the prime mechanisms behind ALS. Mitoxantrone (Mito), a synthetic doxorubicin analog, is an inhibitor of DNA and RNA synthesis/repair via intercalating with nitrogenous bases and inhibiting topoisomerase II. The therapeutic potential of miRNAs associated with disease conditions has also been reported. This study explores the therapeutic potential of Mito along with miRNAs against mutated TDP-43 protein-induced proteinopathy in human-induced pluripotent stem cell (hiPSC)-derived human neural progenitor cells (hNPCs). Methods: HiPSCs mutated for TDP-43 were differentiated into hNPCs and used to explore the therapeutic potential of Mito at a concentration of 1 μM for 24 h (the identified non-cytotoxic dose). The therapeutic effects of Mito on miRNA expression and various cellular parameters such as mitochondrial dynamics, autophagy, and stress granules were assessed using the high-throughput Open Array technique, immunocytochemistry, flow cytometry, immunoblotting, and mitochondrial bioenergetic assay. Results: Mutated TDP-43 protein accumulation causes stress granule formation (G3BP1), mitochondrial bioenergetic dysfunction, SOD1 accumulation, hyperactivated autophagy, and ER stress in hNPCs. The mutated hNPCs also show dysregulation in six miRNAs (miR-543, miR-34a, miR-200c, miR-22, miR-29b, and miR-29c) in mutated hNPCs. A significant restoration of TDP-43 mutation-induced alterations could be witnessed upon the exposure of mutated hNPCs to Mito. Conclusions: Our study indicates that miR-543, miR-29b, miR-22, miR-200c, and miR-34a have antisense therapeutic potential alone and in combination with Mitoxantrone. Full article

The somatic cell count (SCC) is widely used to assess milk quality and diagnose intramammary infections. Several whey proteins have been shown to correlate significantly with SCC and are considered potential indicators of udder health. However, the relationship between milk whey proteins and SCC has not been fully elucidated. In this study, milk samples were grouped into five categories based on SCC levels. Subsequently, whey proteins were identified using a label-free proteomics approach, and the differential abundance of proteins was validated through a selected reaction monitoring (SRM) method. The levels of various proteins, including azurocidin 1 and kininogen-2, exhibited an increase, whereas topoisomerase I, tropomyosin-1, and desmin showed a significant decrease depending on the SCCs. Principal component analysis unveiled that these proteins contributed to the developmental alterations in milk proteins. A majority of these differentially abundant proteins were associated with response to stimulus, localization, and defense response. Our results provide fundamental information on the SCC that can be utilized for evaluating milk quality and serve as potential indicators for detecting intramammary infections. Full article

Cancer remains a leading cause of death worldwide, highlighting the urgent need for novel and more effective treatments. Natural products, with their structural diversity, represent a valuable source for the discovery of anticancer compounds. In this study, we screened 750 Antarctic extracts to identify potential inhibitors of human topoisomerase 1 (hTOP1), a key enzyme in DNA replication and repair, and a target of cancer therapies. Bioassay-guided fractionation led to the identification of palmitic acid (PA) as the active compound from the Antarctic sponge Artemisina plumosa, selectively inhibiting hTOP1. Our results demonstrate that PA irreversibly blocks hTOP1-mediated DNA relaxation and specifically inhibits the DNA religation step of the enzyme’s catalytic cycle. Unlike other fatty acids, PA exhibited unique specificity, which we confirmed through comparisons with linoleic acid. Molecular dynamics simulations and binding assays further suggest that PA interacts with hTOP1-DNA complexes, enhancing the inhibitory effect in the presence of camptothecin (CPT). These findings identify PA as a hTOP1 inhibitor with potential therapeutic implications, offering a distinct mechanism of action that could complement existing cancer therapies. Full article

Glioblastoma (GBM), the most prevalent primary malignant brain tumor, remains challenging to treat due to extensive inter- and intra-tumor heterogeneity. This variability demands combination treatments to improve therapeutic outcomes. A significant obstacle in treating GBM is the expression of O⁶-methylguanine-DNA methyltransferase, a DNA repair enzyme that reduces the efficacy of the standard alkylating agent, temozolomide, in about 50% of patients. This underscores the need for novel, more targeted therapies. Our study investigates the metabolic–epigenetic impact of combining SN-38, a novel topoisomerase inhibitor inducing DNA double-strand breaks, with rabusertib, a checkpoint kinase 1 inhibitor. We identified this synergistic combination through high-throughput drug screening across a panel of GBM cell lines using a cancer drug library combined with SN-38. A secondary metabolic screening with the PEDS algorithm demonstrated a synergistic modulation of purine, one-carbon, and redox metabolism. Furthermore, the combined treatment led to the significant depletion of epigenetically relevant metabolites such as 5-methyl-cytosine, acetyl-lysine, and trimethyl-lysine. Reduced intermediates of the glutathione cycle indicated increased cellular stress following combinatorial treatment. Overall, the combination of SN-38 and rabusertib synergistically disrupts metabolites associated with epigenetic adaptations, leading to cytotoxicity independent of O⁶-methylguanine-DNA methyltransferase status, thereby underpinning this combination as a promising candidate for combinatorial therapy in GBM. Full article

The synthesis of phosphorous indenoquinolines and their biological evaluation as topoisomerase 1 (TOP1) inhibitors and antiproliferative agents were performed. First, the preparation of new hybrid 5H-indeno[2,1-c]quinolines with a phosphine oxide group was performed by a two-step Povarov-type [4+2]-cycloaddition reaction between the corresponding phosphorated aldimines with indene in the presence of BF₃·Et₂O. Subsequent oxidation of the methylene present in the structure resulted in the corresponding indeno[2,1-c]quinolin-7-one phosphine oxides 10. The synthesized derivatives were evaluated as TOP1 inhibitors showing higher inhibition values than CPT at prolonged incubation times (5 min). Inhibition of TOP1 was even observed after 30 min of incubation. The cytotoxic activities of these compounds were also studied against different cancer cell lines and a non-cancerous cell line. While some compounds showed cytotoxicity against some cancerous cells, none of the compounds showed any cytotoxicity against the non-cancerous cell line, MRC-5, in contrast to CPT, which exhibits high toxicity against this cell line. These results represent a very interesting advance since the heterocyclic phosphine oxide derivatives have important properties as TOP1 inhibitors and show an interesting cytotoxicity against different cell lines. Full article

Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are important DNA repair enzymes that remove various adducts from the 3′- and 5′-ends of DNA, respectively. The suppression of the activity of these enzymes is considered as a promising adjuvant therapy for oncological diseases in combination with topoisomerase inhibitors. The simultaneous inhibition of TDP1 and TDP2 may result in greater antitumor effects, as these enzymes can mimic each other’s functions. We have previously shown that usnic acid-based sulfides can act as dual inhibitors, with TDP1 activity in the low micromolar range and their TDP2 at 1 mM. The oxidation of their sulfide moieties to sulfoxides led to an order of magnitude decrease in their cytotoxicity potential, while their TDP1 and TDP2 activity was preserved. In this work, we synthesized new series of usnic acid-based sulfides and their oxidized analogues, i.e., sulfoxides and sulfones, to systematically study these irregularities. The new compounds inhibit TDP1 with IC₅₀ values (the concentration of inhibitor required to reduce enzyme activity by half) in the 0.33–25 μM range. Most sulfides and some sulfoxides and sulfones inhibit TDP2 with an IC₅₀ = 138−421 μM. In addition, the most active compounds synergized (×4) with topotecan on the HeLa cell line as well as causing dose-dependent DNA damage, as confirmed by Comet assay. Sulfides with the 6-methylbenzoimidazol-2-yl substituent (8f, IC₅₀ = 0.33/138 μM, TDP1/2) and sulfones containing a pyridine-2-yl fragment (12k, IC₅₀ = 2/228 μM, TDP1/2) are the most potent derivatives and, therefore, are promising for further development. Full article

Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences (p < 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa. Full article

Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways. Full article

Mycoplasma hyopneumoniae, an important cause of enzootic pneumonia in pigs in many countries, has recently been shown to exhibit reduced susceptibility to several antimicrobial classes. In the present study, a total of 185 pig lung tissue samples were collected from abattoirs in Australia, from which 21 isolates of M. hyopneumoniae were obtained. The antimicrobial resistance profile of the isolates was determined for 12 antimicrobials using minimum inhibitory concentration (MIC) testing, and a subset (n = 14) underwent whole-genome sequence analysis. MIC testing revealed uniformly low values for enrofloxacin (≤1 μg/mL), florfenicol (≤8 μg/mL), lincomycin (≤4 μg/mL), spectinomycin (≤4 μg/mL), tetracycline (≤0.5 μg/mL), tiamulin (≤2 μg/mL), tildipirosin (≤4 μg/mL), tilmicosin (≤16 μg/mL) tulathromycin (≤2 μg/mL), and tylosin (≤2 μg/mL). Higher MICs were observed for erythromycin (MIC range: 16–32 μg/mL), gamithromycin, and tilmicosin (MIC range of both: 32–64 μg/mL). Whole-genome sequencing of the isolates and additional screening using mismatch amplification mutation assay PCR did not identify any known genetic resistance markers within 23S rRNA (macrolides), DNA gyrase A, and topoisomerase IV genes (fluoroquinolones). The WGS data also indicated that the Australian M. hyopneumoniae isolates exhibited limited genetic diversity and formed a distinct monophylectic clade when compared to isolates from other countries. These findings indicate that Australian M. hyopneumoniae likely remains susceptible to the major antimicrobials used to treat enzootic pneumonia in pigs and have evolved in isolation from strains identified in other pig-producing countries. Full article

Dovyalis abyssinica is widely used in Ethiopia for treating various human ailments, yet its pharmacological properties and chemical composition remain largely unexplored. The chromatographic separation of D. abyssinica roots extract afforded five compounds, namely tremulacin (1), cochinchiside A (2), 5-methoxydurmillone (3), catechin-7-O-α-L-rhamnopyranoside (4), and stigmasterol (5), confirmed via IR, NMR, and MS spectral data. This is the first report of these compounds from this plant, except for compounds 1 and 5. The extracts and isolated compounds were tested for antibacterial activity against S. aureus, S. epidermidis, E. faecalis, E. coli, K. pneumoniae, and P. aeruginosa strains. Methanol roots extract exhibited significant antibacterial activity (MIC 0.195 mg/mL) against E. coli and P. aeruginosa. Compounds 1 and 3 showed remarkable antibacterial activity, with compound 1 (MIC 0.625 mg/mL) exhibiting antibacterial activity against S. aureus and S. epidermidis, whereas compound 3 (MIC 0.625 mg/mL) exhibited antibacterial activity against S. epidermidis and K. pneumoniae. Molecular docking analysis revealed better binding energies for compound 1 (−8.0, −9.7, and −8.0 kJ/mol) and compound 3 (−9.0, −8.7, and −8.4 kJ/mol), compared to ciprofloxacin (−8.3, −7.5, and −6.7 kJ/mol), in regard to S. aureus pyruvate kinase, S. epidermidis FtsZ, and K. pneumoniae Topoisomerase IV, respectively. ADME analysis also revealed good antibacterial candidacy of these compounds, provided that in vivo analysis is conducted for further confirmation of the results. Full article