Search Results (158)

Objectives: Lung transplantation (LT) is a rescue therapy for end-stage pulmonary diseases, including systemic autoimmune diseases. The aim of this study was to analyse the evolution of patients with systemic sclerosis (SSc) who, after undergoing LT, become negative for antinuclear antibodies (ANA) and to assess whether they have different clinical and prognostic characteristics than patients who do not become negative. Material and Methods: A retrospective, descriptive analysis was performed over a cohort of patients with a diagnosis of SSc, who underwent unilateral or bilateral LT between 2006 and 2021 at the Vall d’Hebron University Hospital. Clinical and analytical data were obtained from these patients by reviewing their electronic medical records. Two groups of patients were compared: those who tested negative for ANA after LT and those who did not. Statistical analysis was performed with SPSS Statistics 20.0. Results: Eighteen patients were included. The most frequent indication for LT was interstitial lung disease (ILD) combined with pulmonary hypertension (PH), in 13 (72%) patients. All had ANA before the LT (n = 18), and regarding specific SSc autoantibodies, anti-topoisomerase I was presented in 44% (n = 8), anti-U11/U12RNP in 17% (n = 3), anti-RNA Polymerase III in 11.1% (n = 2), anti-Ro52 in 11% (n = 2) and anti-centromere in 6% of individuals (n = 1). 39% (n = 7) of the patients had negative post-LT ANA, 44% (n = 8) had declining titres, and 17% (n = 3) had stable ANA titres. Titres did not increase in any case after LT. Those patients who became ANA-negative after LT were those who had significantly lower titres before LT. No statistically significant differences between groups were found related to pre-LT clinical characteristics, immunosuppressive regimen applied after LT, or in post-LT outcomes. A non-significant trend towards better survival was observed in patients who became ANA negative, with a cumulative survival at 5 years of 85.7% compared to 72.7% among those who remained ANA-positive. Conclusions: Most patients with SSc clear ANA or reduce their levels after LT. A trend towards better survival was observed in this group, compared to the group of transplanted patients who remained positive. Full article

Anthraquinones are molecules with numerous biological properties that can act as DNA intercalators and topoisomerase IIa inhibitors. In this work, the development of technetium-99m radiotracers was pursued via the technetium-tricarbonyl “2 + 1” mixed-ligand approach, fac-[^99mTc][Tc^I(CO)₃(NN′)(N)]⁺, with a (N,N′) bidentate chelator and a N co-ligand. In one approach, the ligands used were 2,2′-bipyridine (bpy) and N-functionalized-imidazole, where imidazole was conjugated to an anthraquinone moiety. In the other approach, 2-picolylamine and imidazole were used as the mixed-ligand system, where picolylamine was conjugated to an anthraquinone moiety. The synthesis of the ligands was achieved by reaction of 2-picolylamine with a suitably functionalized anthraquinone (Aqpa) or anthrapyrazole (Appa) and imidazole with a suitably functionalized anthraquinone (Aqim). The rhenium reference compounds, fac-[Re^I(CO)₃(bpy)(Aqim)]⁺ with bpy as a bidentate chelator and fac-[Re^I(CO)₃(Aqpa or Appa)(Im)]⁺, with imidazole (Im) as a co-ligand, were synthesized and characterized with spectroscopic methods. The radiotracer technetium-99m complexes fac-[^99mTc][Tc(CO)₃(bpy)(Aqim)]⁺ and fac-[^99mTc][Tc(CO)₃(Aqpa or Appa)(Im)]⁺ were prepared and characterized with standard methods. The purified radiotracers displayed high stability (≥90%) after incubation 24 h in 1 mM L-histidine or rat plasma. The tracers’ cell uptake was evaluated in vitro in CT-26 cells, and their pharmacokinetic properties and tumor uptake were evaluated in vivo in CT26-tumor-bearing mice. The “2 + 1” technetium-tricarbonyl approach leads to in vitro stable tracers, and this mixed-ligand system shows promise for further evaluation. 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

Promising protein targets are observed to play a role in multiple pathways across a variety of diseases, such as the regulation of immune responses, cell cycle, senescence, and DNA repair. The oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) can coordinate all these cell characteristics predominately by inhibiting the activity of the serine threonine protein phosphatase 2A (PP2A). CIP2A directly interacts with PP2A and other proteins, such as the DNA damage protein topoisomerase II-binding protein 1, to regulate signal transduction. CIP2A is overexpressed in many human cancers, including small and non-small cell lung cancers. High CIP2A expression in lung cancer correlates with poor prognosis, increased tumor proliferation, and resistance to targeted therapies or chemotherapy. Interestingly, CIP2A expression or signaling is also observed in several non-cancerous pulmonary diseases, such as chronic obstructive pulmonary disease. CIP2A can determine whether DNA-damaged cells enter mitosis and can mediate whether DNA repair occurs. CIP2A is also a regulator of inflammation and possibly fibrotic responses. Its functions are linked to altered NFκB activation and TNFα, IL-1β, IL-4, IL-6, IL-10, IL-13, and TGFβ signaling. This review outlines the possible impact of CIP2A-mediated signaling in pulmonary diseases, the processes that regulate CIP2A responses, CIP2A-dependent pathways, and potential therapeutic strategies targeting CIP2A. Substantial medicinal chemistry efforts are underway to develop therapeutics aimed at modulating CIP2A activity. The development of specific inhibitors of CIP2A that selectively target its expression or protein stability could improve our understanding of CIP2A’s function in pulmonary diseases. Full article

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme for the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, we obtained HEK293A cells with homozygous knockout of the TDP1 gene by the CRISPR/Cas9 method and used them as a cell model to study the mechanisms of anticancer therapy and to investigate the effect of TDP1 gene knockout on gene expression changes in the human HEK293A cell line by transcriptome analysis. In this study, we investigated the effect of a TDP1 inhibitor ((R,E)-2-acetyl-6-(2-(2-(4-bromobenzyliden) hydrazinyl) thiazol-4-yl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d] furan-1(9bH)-one, OL9-119, an usnic acid derivative), capable of potentiating the antitumor effect of topotecan, as well as its combination with topotecan, on the transcriptome of wild-type and TDP1 knockout HEK293A cells. OL9-119 was found to be able to reduce cell motility by decreasing the expression of a number of genes, which may explain the antimetastatic effect of this compound. Differentially expressed genes (DEGs) related to electron transport, mitochondrial function, and protein folding were also identified under TDP1 inhibitor treatment. Full article

Topoisomerase 1 (Top1) removes transcription-related helical torsions and thus plays an important role in preventing genome instability instigated by the formation of non-canonical DNA secondary structures. The genetically tractable Saccharomyces cerevisiae model proved effective in defining the critical function of Top1 to prevent recombination and chromosomal rearrangement at G4-forming genomic loci and studying the human cancer-associated Top1 mutants through the expression of analogous yeast mutants. We previously showed that cleavage-defective Top1 mutants strongly elevate the rate of recombination at G4 DNA, which involves binding to G4 DNA and interaction with the protein nucleolin (Nsr1 in yeast). Here, we further explored the mechanism of genome instability induced by the yeast Top1Y740* mutant, analogous to the human Top1W765Stop mutant conferring resistance to CPT. We show that yTop1Y740* elevates duplications as well as recombination specifically at G4-forming sequences. Interestingly, SUMOylation of yTop1Y740*, which does not affect the G4 DNA-binding or Nsr1-interaction by this mutant, is necessary for such elevated G4-specific genome instability. Many tumors with mutations at the C-terminal residues of Top1, particularly W765, have significantly high G4-associated mutations, underscoring the importance of further investigation into how SUMOylation affects the function of these Top1 mutants at G4 DNA. Full article

Background/Objective: At least 25% of colorectal cancer (CRC) patients develop liver metastases (CRLM), and chemotherapeutic regimens based on the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) provide a survival advantage, but long-term survival is uncommon. The primary molecular target of FP drugs is thymidylate synthase (TS). Methods: A TS/Top1 dual-targeting cytotoxic mechanism for CF10/LV was confirmed by TS ternary complex detection by Western blot and by immunofluorescence detection of Top1 cleavage complexes. CF10/LV activated the ATR/Chk1 pathway consistent with enhanced replication stress and induced apoptosis. In vivo studies showed CF10 and CF10/LV eradicated liver metastasis in a CRLM model without scarring or weight loss, displaying therapeutic advantages relative to legacy FPs. Results: We demonstrated that a nanoscale FP polymer, CF10, displayed greater potency than expected based on FP content in part through more direct conversion to the TS-inhibitory metabolite, FdUMP. In this study, we tested CF10 for potency advantages relative to 5-FU and trifluorothymidine (TFT, the FP component of TAS-102) and confirmed a general potency advantage for CF10 in CRC cell lines in the Broad Institute PRISM screen. We demonstrated that this potency advantage is retained in CRC cells cultured with human-like folate levels and is enhanced by LV co-treatment to a similar extent as that by 5-FU. Our results confirm CF10 development proceeding as a CF10/LV combination. Mechanistically, CF10 cytotoxicity closely correlates with poisons of DNA topoisomerase 1 (Top1) in the PRISM screen relative to 5-FU and TFT. Conclusions: Our pre-clinical data support an early-phase clinical trial for CF10 for treating liver-metastatic CRC. Full article

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