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Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver condition worldwide, demanding further investigation into its pathogenesis. Circular RNAs (circRNAs) are emerging as pivotal regulators in MASLD processes, yet their pathological implications in MASLD remain poorly understood. This study focused on elucidating the role of circular RNA ribonucleotide reductase subunit M2 (circRRM2) in MASLD progression. In this study, we used both in vitro and in vivo MASLD models using long-chain-free fatty acid (FFA)-treated hepatocytes and high-fat diet (HFD)-induced MASLD in mice, respectively. We determined the expression patterns of circRRM2, microRNA-142-5p (miR-142-5p), and neuregulin 1 (NRG1) in livers of MASLD-afflicted mice and MASLD hepatocytes by RT-qPCR. Dual-luciferase reporter assays verified the binding relationships among circRRM2, miR-142-5p, and NRG1. We conducted further analyses of their roles in MASLD hepatocytes and modulated circRRM2, miR-142-5p, and NRG1 expression in vitro by transfection. Our findings were validated in vivo. The results demonstrated reduced levels of circRRM2 and NRG1, along with elevated miR-142-5p expression in MASLD livers and hepatocytes. Overexpression of circRRM2 downregulated lipogenesis-related genes and decreased triglycerides accumulation in livers of MASLD mice. MiR-142-5p, which interacts with circRRM2, effectively counteracted the effects of circRRM2 in MASLD hepatocytes. Furthermore, NRG1 was identified as a miR-142-5p target, and its overexpression mitigated the regulatory impact of miR-142-5p on MASLD hepatocytes. In conclusion, circRRM2, via its role as a miR-142-5p sponge, upregulating NRG1, possibly influenced triglycerides accumulation in both in vitro and in vivo MASLD models. NEW & NOTEWORTHY: CircRRM2 expression was downregulated in free fatty acid (FFA)-challenged hepatocytes and high-fat diet (HFD) fed mice. Overexpressed circular RNA ribonucleotide reductase subunit M2 (circRRM2) attenuated metabolic dysfunction-associated steatotic liver disease (MASLD) development by suppressing FFA-induced triglycerides accumulation. CircRRM2 targeted microRNA-142-5p (miR-142-5p), which served as an upstream inhibitor of neuregulin 1 (NRG1) and collaboratively regulated MASLD progression. [ABSTRACT FROM AUTHOR]

Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae. Author summary: Zn and Mn are essential micronutrients for many bacteria, including Streptococcus pneumoniae. While Zn performs vital structural or catalytic roles in certain proteins, in excess, Zn can inhibit Mn uptake by S. pneumoniae and displace, but not functionally replace Mn from key enzymes including superoxide dismutase A (SodA). Here, we show that the Ccn small regulatory RNAs promote S. pneumoniae resistance to Zn intoxication. Furthermore, we demonstrate that these small regulatory RNAs modulate the ability of S. pneumoniae to cause invasive pneumonia. Altogether, these findings reveal a new layer of regulation of S. pneumoniae Zn homeostasis and suggest that there are factors in addition to known transporters that modulate intracellular, bioavailable Zn levels. [ABSTRACT FROM AUTHOR]

Gallium-based therapy has been considered a potentially effective cancer therapy for decades and has recently re-emerged as a novel therapeutic strategy for the management of glioblastoma tumors. Gallium targets the iron-dependent phenotype associated with aggressive tumors by mimicking iron in circulation and gaining intracellular access through transferrin-receptor-mediated endocytosis. Mechanistically, it is believed that gallium inhibits critical iron-dependent enzymes like ribonucleotide reductase and NADH dehydrogenase (electron transport chain complex I) by replacing iron and removing the ability to transfer electrons through the protein secondary structure. However, information regarding the effects of gallium on cellular iron metabolism is limited. As mitochondrial iron metabolism serves as a central hub of the iron metabolic network, the goal of this study was to investigate the effects of gallium on mitochondrial iron metabolism in glioblastoma cells. Here, it has been discovered that gallium nitrate can induce mitochondrial iron depletion, which is associated with the induction of DNA damage. Moreover, the generation of gallium-resistant cell lines reveals a highly unstable phenotype characterized by impaired colony formation associated with a significant decrease in mitochondrial iron content and loss of the mitochondrial iron uptake transporter, mitoferrin-1. Moreover, gallium-resistant cell lines are significantly more sensitive to radiation and have an impaired ability to repair any sublethal damage and to survive potentially lethal radiation damage when left for 24 h following radiation. These results support the hypothesis that gallium can disrupt mitochondrial iron metabolism and serve as a potential radiosensitizer. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) infection can exacerbate liver disease progression through multiple mechanisms, eventually leading to hepatocellular carcinoma (HCC). HBV-encoded oncogene X protein (HBx), a key regulatory protein of HBV infection, serves as a positive regulator of hepatocarcinogenesis. The indispensability of the M2 subunit of ribonucleotide-diphosphate reductase (RRM2) lies in its role in facilitating DNA replication and repair processes. In our previous investigation, it was postulated that the gene RRM2 exhibits elevated expression levels in several categories of malignant tumors, particularly in HBV-related HCC. Additionally, it was observed that RRM2 is present within protein complexes that are centered on HBx. In the present investigation, the objective of this work was to investigate the potential relationship between the elevated expression of RRM2 in HBV-related HCC and the influence of HBx on this expression. The study attempted to determine the specific mechanism by which RRM2 is implicated in the promotion of hepatocarcinogenesis by HBx. There have been multiple scholarly proposals suggesting that the induction of autophagy by HBx is a significant intermediary factor in the development of HCC. However, the precise carcinogenic function of HBx-induced autophagy remains a subject of debate. Results: This work initially investigated the impact of suppressing cellular autophagy on the malignant biological behaviors of HBx-promoted cells using an in vitro cellular model. The findings revealed that the suppression of cellular autophagy partially disrupted the oncogenic effects of HBx. In light of this, we proceeded to conduct more investigations into the regulatory association between RRM2 and HBx-induced autophagy in the upstream-downstream context. Our data indicate that HBx proteins increase the expression of RRM2. Suppression of RRM2 expression not only hinders HBx-induced autophagy, but also worsens the cellular G1/S blockage and reduces the HBx-induced malignant growth of hepatocellular carcinoma tumors, while stimulating apoptosis. Conclusions: Therefore, we hypothesised that RRM2 is a potential downstream target of HBx-induced hepatocarcinogenesis, and mining the oncogenic mechanism of RRM2 is significant in exploring the preventive treatment of HBV-related HCC. [ABSTRACT FROM AUTHOR]

This article presents a deep-learning model that uses RNA-seq data to predict the chemoresistance of platinum-based therapy in high-grade serous ovarian carcinoma (HGSOC). The model, which was developed using diverse transcriptome datasets and deep ensemble learning techniques, achieved high performance in predicting chemo-resistant HGSOC patients. The study highlights the importance of accurate prediction methods to guide treatment decisions and improve patient outcomes in HGSOC, a disease with high mortality rates worldwide. The identified genes and prediction models have potential biological relevance and may be useful for further research and application in other ovarian cancer studies with limited sample sizes. [Extracted from the article]

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism. [ABSTRACT FROM AUTHOR]

Background: Genetic polymorphisms of molecules are known to cause individual differences in the therapeutic efficacy of anticancer drugs. However, to date, germline mutations (but not somatic mutations) for anticancer drugs have not been adequately studied. The objective of this study was to investigate the association between germline polymorphisms of gemcitabine metabolic and transporter genes with carbohydrate antigen 19‐9 (CA 19‐9) response (decrease ≥50% from the pretreatment level at 8 weeks) and overall survival (OS) in patients with metastatic pancreatic cancer who receive gemcitabine‐based chemotherapy. Methods: This multicenter, prospective, observational study enrolled patients with metastatic pancreatic cancer patients who were receiving gemcitabine monotherapy or gemcitabine plus nanoparticle albumin‐bound paclitaxel combination chemotherapy. Thirteen polymorphisms that may be involved in gemcitabine responsiveness were genotyped, and univariate and multivariate logistic regression analyses were used to determine the association of these genotypes with CA 19‐9 response and OS. The significance level was set at 5%. Results: In total, 180 patients from 11 hospitals in Japan were registered, and 159 patients whose CA 19‐9 response could be assessed were included in the final analysis. Patients who had a CA 19‐9 response had significantly longer OS (372 vs. 241 days; p =.007). RRM1 2464A>G and RRM2 175T>G polymorphisms suggested a weak association with CA 19‐9 response and OS, but it was not statistically significant. COX‐2 −765G>C polymorphism did not significantly correlate with CA 19‐9 response but was significantly associated with OS (hazard ratio, 2.031; p =.019). Conclusions: Genetic polymorphisms from the pharmacokinetics of gemcitabine did not indicate a significant association with efficacy, but COX‐2 polymorphisms involved in tumor cell proliferation might affect OS. This study investigated the association between germline polymorphisms of gemcitabine metabolic and transporter genes and carbohydrate 19‐9 response and overall survival in patients with metastatic pancreatic cancer who were receiving gemcitabine‐based chemotherapy. Germline polymorphisms of the metabolic pathway and transporter genes in gemcitabine were not significantly associated with efficacy, but COX‐2 polymorphisms were associated with overall survival. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1. [ABSTRACT FROM AUTHOR]

Hydroxyurea (HU; hydroxycarbamide) is a chemotherapy medication used to treat various types of cancer and other diseases such as sickle cell anemia. HU inhibits DNA synthesis by targeting ribonucleotide reductase (RNR). Recent studies have suggested that HU also causes oxidative stress in living systems. In the present study, we investigated if HU could directly affect the activity and/or conformation of DNA. We measured in vitro gene expression in the presence of HU by adapting a cell-free luciferase assay. HU exhibited a bimodal effect on gene expression, where promotion or inhibition were observed at lower or higher concentrations (mM range), respectively. Using atomic force microscopy (AFM), the higher-order structure of DNA was revealed to be partially-thick with kinked-branching structures after HU was added. An elongated coil conformation was observed by AFM in the absence of HU. Single DNA molecules in bulk aqueous solution under fluctuating Brownian motion were imaged by fluorescence microscopy (FM). Both spring and damping constants, mechanical properties of DNA, increased when HU was added. These experimental investigations indicate that HU directly interacts with DNA and provide new insights into how HU acts as a chemotherapeutic agent and targets other diseases. [ABSTRACT FROM AUTHOR]

Recently, there has been great interest in plant-derived compounds known as phytochemicals. The pentacyclic oleanane-, ursane-, and lupane-type triterpenes are phytochemicals that exert significant activity against diseases like cancer. Lung cancer is the leading cause of cancer-related death worldwide. Although chemotherapy is the treatment of choice for lung cancer, its effectiveness is hampered by the dose-limiting toxic effects and chemoresistance. Herein, we investigated six pentacyclic triterpenes, oleanolic acid, ursolic acid, asiatic acid, betulinic acid, betulin, and lupeol, on NSCLC A549 cells. These triterpenes have several structural variations that can influence the activation/inactivation of key cellular pathways. From our results, we determined that most of these triterpenes induced apoptosis, S-phase and G2/M-phase cycle arrest, the downregulation of ribonucleotide reductase (RR), reactive oxygen species, and caspase 3 activation. For chemoresistance markers, we found that most triterpenes downregulated the expression of MAPK/PI3K, STAT3, and PDL1. In contrast, UrA and AsA also induced DNA damage and autophagy. Then, we theoretically determined other possible molecular targets of these triterpenes using the online database ChEMBL. The results showed that even slight structural changes in these triterpenes can influence the cellular response. This study opens up promising perspectives for further research on the pharmaceutical role of phytochemical triterpenoids. [ABSTRACT FROM AUTHOR]

N-Hydroxyurea (HU) is an important chemotherapeutic agent used as a first-line treatment in conditions such as sickle cell disease and β -thalassemia, among others. To date, its properties as a hydrated molecule in the blood plasma or cytoplasm are dramatically understudied, although they may be crucial to the binding of HU to the radical catalytic site of ribonucleotide reductase, its molecular target. The purpose of this work is the comprehensive exploration of HU hydration. The topic is studied using ab initio molecular dynamic (AIMD) simulations that apply a first principles representation of the electron density of the system. This allows for the calculation of infrared spectra, which may be decomposed spatially to better capture the spectral signatures of solute–solvent interactions. The studied molecule is found to be strongly hydrated and tightly bound to the first shell water molecules. The analysis of the distance-dependent spectra of HU shows that the E and Z conformers spectrally affect, on average, 3.4 and 2.5 of the closest H2O molecules, respectively, in spheres of radii of 3.7 Å and 3.5 Å, respectively. The distance-dependent spectra corresponding to these cutoff radii show increased absorbance in the red-shifted part of the water OH stretching vibration band, indicating local enhancement of the solvent's hydrogen bond network. The radially resolved IR spectra also demonstrate that HU effortlessly incorporates into the hydrogen bond network of water and has an enhancing effect on this network. Metadynamics simulations based on AIMD methodology provide a picture of the conformational equilibria of HU in solution. Contrary to previous investigations of an isolated HU molecule in the gas phase, the Z conformer of HU is found here to be more stable by 17.4 kJ·mol−1 than the E conformer, pointing at the crucial role that hydration plays in determining the conformational stability of solutes. The potential energy surface for the OH group rotation in HU indicates that there is no intramolecular hydrogen bond in Z-HU in water, in stark contrast to the isolated solute in the gas phase. Instead, the preferred orientation of the hydroxyl group is perpendicular to the molecular plane of the solute. In view of the known chaotropic effect of urea and its N-alkyl-substituted derivatives, N-hydroxyurea emerges as a unique urea derivative that exhibits a kosmotropic ordering of nearby water. This property may be of crucial importance for its binding to the catalytic site of ribonucleotide reductase with a concomitant displacement of a water molecule. [ABSTRACT FROM AUTHOR]

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65–229 of DHODH and the amino acids 25–40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV. [ABSTRACT FROM AUTHOR]

The Bis-(1-(2,4-dichlorobenzoyl)-3-methylthiourea) iron (III) complex has been synthesized from the reaction between 1-(2,4-dichlorobenzoyl)-3- methylthiourea and Fe (III) metal ion by reflux method with ethanol solvent at a temperature of 75°C for 7 hours. It was characterized by a hot stage microscope (HSM), UV-Vis, FT-IR, and mass spectroscopy. The % yield of the synthesis result was 97.58%. From the docking study on the ribonucleotide reductase enzyme, the binding affinity value was -7.76 kcal/mol, and the inhibition constant was 2.11 mM. The Bis-(1-(2,4-dichlorobenzoyl)-3 methylthiourea) iron (III) complex compounds can be synthesized and predicted as anticancer candidates. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a common inflammatory and autoimmune disease. Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) is a crucial and a rate-limiting enzyme responsible for deoxynucleotide triphosphate(dNTP) production. We have found a high expression level of RRM2 in patients with RA, but the molecular mechanism of its action remains unclear. Methods: We analyzed the expression of hub genes in RA using GSE77298 datasets downloaded from Gene Expression Omnibus database. RRM2 and insulin-like growth factor-2 messenger ribonucleic acid (mRNA)-binding protein 3 (IGF2BP3) gene knockdown was achieved by infection with lentiviruses. The expression of RRM2, IGF2BP3, matrix metalloproteinase (MMP)-1, and MMP-9 were detected via western blotting assay. Cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MeRIP-qRT-PCR was performed to test the interaction of IGF2BP3 and RRM2 mRNA via m6A modification. Cell proliferation was determined by clone formation assay. Migration and invasion assays were performed using transwell Boyden chamber. Results: RRM2 and IGF2BP3 were highly expressed in clinical specimens and tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β-stimulated synovial cells. RRM2 and IGF2BP3 knockdown inhibited the proliferation, migration, and invasion of MH7A cells. The inhibitory effects of IGF2BP3 knockdown were effectively reversed by simultaneously overexpressing RRM2 in MH7A cells. By analyzing N6-methyladenosine (m6A)2Target database, five m6A regulatory target binding sites for IGF2BP3 were identified in RRM2 mRNA, suggesting a direct relationship between IGF2BP3 and RRM2 mRNA. Additionally, in RRM2 small hairpin (sh)RNA lentivirus-infected cells, the levels of phosphorylated Akt and MMP-9 were significantly decreased compared with control shRNA lentivirus-infected cells. Conclusion: The present study demonstrated that RRM2 promoted the Akt phosphorylation leading to high expression of MMP-9 to promote the migration and invasive capacities of MH7A cells. Overall, IGF2BP promotes the expression of RRM2, and regulates the migration and invasion of MH7A cells via Akt/MMP-9 pathway to promote RA progression. [ABSTRACT FROM AUTHOR]

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity. The clinical application of inhibitors targeting checkpoint kinase 1 (CHK1) is challenged by limited efficacy. Here, the authors identify that thioredoxin (Trx) system inhibition mediates sensitivity to CHK1 inhibitor via regulating the activity of ribonucleotide reductase, demonstrating the synergistic effect of CHK1 inhibitor and inhibitors targeting Trx system in lung cancer models. [ABSTRACT FROM AUTHOR]

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 –but not RPA–from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis. Author summary: Cells have to accurately replicate their genomes to avoid loss of genetic information during cell divisions. This is achieved by coordinating the advance of the replication machinery with different mechanisms that prevent and/or repair errors during DNA synthesis. However, different DNA lesions accumulate during DNA replication that are repaired at DNA repair centers by error-free homologous recombination and error-prone translesion synthesis mechanisms. Here, we show that the replicative helicase and the ribonucleotide reductase complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication interact with each other in a DNA damage-regulated manner. Furthermore, we observe that these interactions facilitate the dynamics of a key recombination protein (Rad52) at the repair centers and prevent genetic instability by translesion synthesis through mechanisms that are not associated with the canonical functions of the replicative helicase and the ribonucleotide reductase complexes. We propose that these two essential complexes play non-replicative roles in preventing genetic instability in response to DNA damage by genetically regulated physical interactions. [ABSTRACT FROM AUTHOR]

Azido-modified nucleosides have been extensively explored as substrates for click chemistry and the metabolic labeling of DNA and RNA. These compounds are also of interest as precursors for further synthetic elaboration and as therapeutic agents. This review discusses the chemistry of azidonucleosides related to the generation of nitrogen-centered radicals (NCRs) from the azido groups that are selectively inserted into the nucleoside frame along with the subsequent chemistry and biological implications of NCRs. For instance, the critical role of the sulfinylimine radical generated during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxy pyrimidine nucleotides as well as the NCRs generated from azidonucleosides by radiation-produced (prehydrated and aqueous) electrons are discussed. Regio and stereoselectivity of incorporation of an azido group ("radical arm") into the frame of nucleoside and selective generation of NCRs under reductive conditions, which often produce the same radical species that are observed upon ionization events due to radiation and/or other oxidative conditions that are emphasized. NCRs generated from nucleoside-modified precursors other than azidonucleosides are also discussed but only with the direct relation to the same/similar NCRs derived from azidonucleosides. [ABSTRACT FROM AUTHOR]

This study investigated the meat quality, expression of myosin heavy chain (MyHC) and metabolism-related genes, ribonucleotides and fatty acids in Longissimus thoracis of Thai native pigs (TNPs) from different geographical regions (GR). Forty-one 9–10-month-old castrated TNPs (BW 60 kg), consisting of 18, 11 and 12 pigs from Northern (NT), Southern (ST) and Northeastern (NE) regions, respectively, were slaughtered. GR did not affect (p > 0.05) the expression of MyHC, phosphoglycerate mutase 1, cytosolic glycerol-3-phosphate dehydrogenase, triosephosphate isomerase 1 and adipocyte fatty acid binding protein genes. The trend of MyHC was MyHC IIx > MyHC IIb > MyHC IIa > MyHC I. The NT loin had higher (p < 0.05) glycogen, C18:2n6, C20:4n6 and cooking loss, lower inosine, inosine monophosphate and hypoxanthine and a shorter sarcomere length than the ST and NE loins. The ST loin had a lower (p < 0.05) a* compared to other loins. Principal component analysis established significant relationships between the TNP and specific meat quality traits. This finding suggests that GR affected the meat quality, ribonucleotides and selected fatty acids in TNPs. These results provide relevant information that can be used to optimize the use of Thai native pork. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a systemic immune-related disease characterized by synovial inflammation and destruction of joint cartilage. The pathogenesis of RA remains unclear, and diagnostic markers with high sensitivity and specificity are needed urgently. This study aims to identify potential biomarkers in the synovium for diagnosing RA and to investigate their association with immune infiltration. Methods: We downloaded four datasets containing 51 RA and 36 healthy synovium samples from the Gene Expression Omnibus database.Differentially expressed genes were identified using R. Then, various enrichment analyses were conducted. Subsequently, weighted gene co-expression network analysis (WGCNA), random forest (RF), support vector machine--recursive feature elimination (SVM-RFE), and least absolute shrinkage and selection operator (LASSO)were used to identify the hub genes for RA diagnosis. Receiver operating characteristic curves and nomogram models were used to validate the specificity and sensitivity of hub genes. Additionally, we analyzed the infiltration levels of 28 immune cells in the expression profile and their relationship with the hub genes using single-sample gene set enrichment analysis. Results: Three hub genes, namely, ribonucleotide reductase regulatory subunit M2 (RRM2), DLG-associated protein 5 (DLGAP5), and kinesin family member 11 (KIF11), were identified through WGCNA, LASSO, SVM-RFE, and RF algorithms. These hub genes correlated strongly with T cells, natural killer cells, and macrophage cells as indicated by immune cell infiltration analysis. Conclusion: RRM2, DLGAP5, and KIF11 could serve as potential diagnostic indicators and treatment targets for RA. The infiltration of immune cells offers additional insights into the underlying mechanisms involved in the progression of RA. [ABSTRACT FROM AUTHOR]

It is well known that the adaptations of muscular antioxidant system to aerobic exercise depend on the frequency, intensity, duration, type of the exercise. Nonetheless, the timing of aerobic exercise, related to circadian rhythms or biological clock, may also affect the antioxidant defense system, but its impact remains uncertain. Bain and muscle ARNT-like 1 (BMAL1) is the core orchestrator of molecular clock, which can maintain cellular redox homeostasis by directly controlling the transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2). So, our research objective was to evaluate the impacts of aerobic exercise training at various time points of the day on BMAL1 and NRF2-mediated antioxidant system in skeletal muscle. C57BL/6J mice were assigned to the control group, the group exercising at Zeitgeber Time 12 (ZT12), and the group exercising at ZT24. Control mice were not intervened, while ZT12 and ZT24 mice were trained for four weeks at the early and late time point of their active phase, respectively. We observed that the skeletal muscle of ZT12 mice exhibited higher total antioxidant capacity and lower reactive oxygen species compared to ZT24 mice. Furthermore, ZT12 mice improved the colocalization of BMAL1 with nucleus, the protein expression of BMAL1, NRF2, NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate-cysteine ligase modifier subunit and glutathione reductase in comparison to those of ZT24 mice. In conclusion, the 4-week aerobic training performed at ZT12 is more effective for enhancing NRF2-mediated antioxidant responses of skeletal muscle, which may be attributed to the specific activation of BMAL1. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2′, 2′-difluoro 2′deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2 , in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aβ1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200 ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200 ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment. [ABSTRACT FROM AUTHOR]

Background: Vanillin is a flavoring substance derived from vanilla. We are currently developing a biotransformation method for vanillin production using glucose. This report describes the last step in vanillin production: the conversion of vanillic acid to vanillin. First, we selected Corynebacterium glutamicum as the host owing to its high vanillin resistance. The aromatic aldehyde reductase gene (NCgl0324) and vanillic acid demethylase protein subunits A and B gene (vanAB, NCgl2300-NCgl2301) were deleted in C. glutamicum genome to avoid vanillin degradation. Next, we searched for an aromatic carboxylic acid reductase (ACAR), which converts vanillic acid to vanillin. Seventeen ACAR homologs from various organisms were introduced into C. glutamicum. Results: In vivo conversion experiments showed that eight ACARs were successfully expressed and produced vanillin. In terms of conversion activity and substrate specificity, the ACARs from Gordonia effusa, Coccomyxa subellipsoidea, and Novosphingobium malaysiense are promising candidates for commercial production. Conclusions: Corynebacterium glutamicum harboring Gordonia effusa ACAR produced 22 g/L vanillin, which is, to the best of our knowledge, the highest accumulation reported in the literature. At the same time, we discovered ACAR from Novosphingobium malaysiense and Coccomyxa subellipsoidea C-169 with high substrate specificity. These findings are useful for reducing the byproducts. [ABSTRACT FROM AUTHOR]

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3′-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3′-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3′-rA:T and 3′-rG:C step 3 structures with step 2 structures of canonical 3′-dA:T and 3′-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2′-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3′-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair. [ABSTRACT FROM AUTHOR]

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes. [ABSTRACT FROM AUTHOR]

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes. This study reports the direct detection of ribonucleotides embedded in DNA molecules by Oxford Nanopore sequencing, based on ribonucleotides-induced alterations in basecalling features, currents, and dwell times. [ABSTRACT FROM AUTHOR]

Background: Although there is evidence that ribonucleotide reductase subunit M2 (RRM2) is associated with numerous cancers, pan-cancer analysis has seldom been conducted. This study aimed to explore the potential carcinogenesis of RRM2 in pan-cancer using datasets from The Cancer Genome Atlas (TCGA). Methods: Data from the UCSC Xena database were analyzed to investigate the differential expression of RRM2 across multiple cancer types. Clinical data such as age, race, sex, tumor stage, and status were acquired to analyze the influence of RRM2 on the clinical characteristics of the patients. The role of RRM2 in the onset and progression of multiple cancers has been examined in terms of genetic changes at the molecular level, including tumor mutational burden (TMB), microsatellite instability (MSI), biological pathway changes, and the immune microenvironment. Results: RRM2 was highly expressed in most cancers, and there was an obvious correlation between RRM2 expression and patient prognosis. RRM2 expression is associated with the infiltration of diverse immune and endothelial cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI). Moreover, the cell cycle is involved in the functional mechanisms of RRM2. Conclusions: Our pan-cancer study provides a comprehensive understanding of the carcinogenesis of RRM2 in various tumors. [ABSTRACT FROM AUTHOR]

Smart gene delivery vectors are gaining increasing attention in gene therapy, owing to their low cytotoxicity and intrinsic responsiveness. Our previously fabricated novel cationic brush polymer, comprising C Se bonds and tertiary amine EGIn-g-PDMAEMA, shows potential for gene transfection. In this study, its high efficiency for siRNA/pDNA transfection and low cytotoxicity in reactive oxygen species (ROS)-rich microenvironments is substantiated in vitro. Its superior binding capacity with siRNA/pDNA is confirmed by agarose gel electrophoresis assay. The threshold weight ratios for siRNA/pDNA migration delay are 15 and 3 (polymer-to-nucleic acid, w/w), respectively. Fluorescence microscopy and ribonucleotide reductase regulatory subunit M2 gene silencing essay verify the biodegradability and responsive control release of nucleic acids under hydrogen peroxide stimulation in Huh-7 cells. Compared with the gold standard, polyethylenimine 25 kDa, the target polymer displays superior transfection efficiency in ROS-rich tumor cells under serumfree conditions. Furthermore, the vector-nucleic acid complexes exhibit over 90% cell viability at a high concentration of 12 µg mL-1 and good colloidal stability in phosphate-buffered saline (PBS) and 10% fetal bovine serum-PBS for 24 h. The efficient control release and expression of nucleic acids in ROS environments and reduced cytotoxicity highlight the superiority of EGIn-g- PDMAEMA as a gene delivery platform for tumor gene therapy. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of this study was to investigate the roles of ribonucleotide reductase subunit M2 (RRM2; subunit of ribonucleotide reductase) in severe woody breast (WB) and normal breast muscles. ANIMALS: 40 8-week-old male Ross-708 broiler chickens. METHODS: Quantitative PCR was performed to determine gene expression, and commercial ELISA/assay kits were used to obtain several enzymatic activities. RESULTS: Results showed that RRM2 activity (P = .0002) and RRM2 (P = .05) and hydroxymethylbilane synthase expression (impaired oxygen transport and metabolism, P = .002) were reduced in WB, while caveolin-3 (defected membrane integrity, P = .09), endoglin (increased fibrosis, P = .06), and secreted protein acidic rich in cysteine (metabolic dysregulation, P = .09) expression tended to increase in WB. WB tended to have increased levels of homocysteine (P = .06), aspartate aminotransferase mitochondria (P = .02), pyruvate kinase (P = .04), DNA damage (P = .06), creatine kinase (P = .05), and triglyceride (P = .002) but decreased ATPase activity (P = .01), all indicating mitochondria dysfunction and tissue damage. CLINICAL RELEVANCE: In this study, differences in various enzyme activities and increased DNA damage suggest that RRM2-mediated mitochondrial abnormalities may play a role in WB myopathy. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa. [ABSTRACT FROM AUTHOR]

Mitochondria undergo fission and fusion that are critical for cell survival and cancer development, while the regulatory factors for mitochondrial dynamics remain elusive. Herein we found that RNA m6A accelerated mitochondria fusion of colorectal cancer (CRC) cells. Metabolomics analysis and function studies indicated that m6A triggered the generation of glutathione (GSH) via the upregulation of RRM2B—a p53-inducible ribonucleotide reductase subunit with anti-reactive oxygen species potential. This in turn resulted in the mitochondria fusion of CRC cells. Mechanistically, m6A methylation of A1240 at 3′UTR of RRM2B increased its mRNA stability via binding with IGF2BP2. Similarly, m6A methylation of A2212 at the coding sequence (CDS) of OPA1—an essential GTPase protein for mitochondrial inner membrane fusion—also increased mRNA stability and triggered mitochondria fusion. Targeting m6A through the methyltransferase inhibitor STM2457 or the dm6ACRISPR system significantly suppressed mitochondria fusion. In vivo and clinical data confirmed the positive roles of the m6A/mitochondrial dynamics in tumor growth and CRC progression. Collectively, m6A promoted mitochondria fusion via induction of GSH synthesis and OPA1 expression, which facilitated cancer cell growth and CRC development. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic cancer (PC) remains a lethal disease, and gemcitabine resistance is prevalent. However, the biomarkers suggestive of gemcitabine resistance remain unclear. Methods: Bioinformatic tools identified ribonucleotide reductase catalytic subunit M1 (RRM1) in gemcitabine-related datasets. A cox regression model revealed the predictive value of RRM1 with clinical features. An external clinical cohort confirmed the prognostic value of RRM1. RRM1 expression was validated in gemcitabine-resistant cells in vitro and in orthotopic PC model. CCK8, flow cytometry, transwell migration, and invasion assays were used to explore the effect of RRM1 on gemcitabine-resistant cells. The CIBERSORT algorithm investigated the impact of RRM1 on immune infiltration. Results: The constructed nomogram based on RRM1 effectively predicted prognosis and was further validated. Moreover, patients with higher RRM1 had shorter overall survival. RRM1 expression was significantly higher in PC tissue and gemcitabine-resistant cells in vitro and in vivo. RRM1 knockdown reversed gemcitabine resistance, inhibited migration and invasion. The infiltration levels of CD4 + T cells, CD8 + T cells, neutrophils, and plasma cells correlated markedly with RRM1 expression, and communication between tumor and immune cells probably depends on NF-κB/mTOR signaling. Conclusion: RRM1 may be a potential marker for prognosis and a target marker for gemcitabine resistance in PC. [ABSTRACT FROM AUTHOR]

DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells. Ribonucleotides are incorporated into DNA during every S phase. Here, the authors show that replisome protein RTF2 localizes RNase H2 to the replisome, promoting ribonucleotide removal by RNase H2 when replication is ongoing but interfering with PRIM1-dependent restart following fork stalling. [ABSTRACT FROM AUTHOR]

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L−1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone. Ipatasertib is a potent Akt (protein kinase B) inhibitor synthesized via a chemoenzymatic process. Here, the authors use mutational scanning and algorithm-aided enzyme engineering to optimize a ketoreductase from Sporidiobolus salmonicolor and generate a 10-amino acid substituted variant exhibiting a 64-fold higher kcat and improved yield for the relevant alcohol intermediate, with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L−1 ketone after 30 h. [ABSTRACT FROM AUTHOR]

Globally, the occurrence of skin cancers has been increasing day by day due to unprotected skin and exposure to UV radiation. This research is focused toward the evaluation of the antioxidant potential of an OH-free flavanone derivative that was synthesized by using 1,4-diazabicyclo[2.2.2]octane–polyethylene glycol (DABCO-PEG) 400. Ionic liquid was prepared via the alkylation of DABCO using 1-pentyl bromide followed by mixing with PEG 400. The structure of the synthesized molecules was characterized through single-crystal XRD. The target flavanone, viz., 2-(4-isobutylphenyl)chroman-4-one, was subjected to free radical activity. In addition, in silico studies were carried out with proteins ribonucleotide reductase and tyrosinase and isobutyl containing flavanone, viz., 2-(4-isobutylphenyl)chroman-4-one. The flavanone 2-(4-isobutylphenyl)chroman-4-one showed significant inhibition at a concentration of 25 μg/ml compared to vitamin C, which was also supported by the molecular docking studies. The flavanones exhibit binding energies of −6.45 and −6.83 kcal/mol for ribonucleotide reductase and tyrosinase, respectively. The results were further validated by molecular dynamic simulations, which recommended that further investigation of this flavanone must be carried out before using it in potent drug discovery in the field of skin cancer. [ABSTRACT FROM AUTHOR]

Maintaining a balanced redox state within cells is crucial for the sustenance of life. The process involves continuous cytosolic disulfide reduction reactions to restore oxidized proteins to their reduced thiol forms. There are two main cellular antioxidant pathways—the thioredoxin (Trx) and glutathione (GSH)/glutaredoxin (Grx) systems. In the GSH/Grx system, glutathione reductase (GR; GSR) catalyses the reduction of GSH disulfide (GSSG) to its sulfhydryl form (GSH), which can then further reduce oxidized Grxs. GR is an essential enzyme that helps in maintaining the supply of reduced glutathione-GSH, which is a significant reducing thiol found in most cells and known for its antioxidant properties. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of GR protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of GR and tumour histological grade, depth of invasion, regional lymph node involvement, staging, and PCNA immunohistochemical expression. It was found that 95% of patients with stage I had low levels of GR expression, whereas 89% of patients with stage III had high levels of immunohistochemical expression. A high level of expression was also detected in the patients with stage II of the disease, where almost 63% were characterized by a high expression of GR. The Western blot method revealed that the highest level of expression was found in the LS 174T cell line, which corresponds to stage II. The results of our study indicate that the immunohistochemical expression of GR may act as an independent prognostic factor associated with colon adenocarcinoma patients' prognosis. [ABSTRACT FROM AUTHOR]

Background: Atypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT. Methods: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT. Results: RRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells. Conclusions: Collectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT. [ABSTRACT FROM AUTHOR]

One fundamental principle that underlies various cancer treatments, such as traditional chemotherapy and radiotherapy, involves the induction of catastrophic DNA damage, leading to the apoptosis of cancer cells. In our study, we conduct a comprehensive dose-response combination screening focused on inhibitors that target key kinases involved in the DNA damage response (DDR): ATR, ATM, and DNA-PK. This screening involves 87 anti-cancer agents, including six DDR inhibitors, and encompasses 62 different cell lines spanning 12 types of tumors, resulting in a total of 17,912 combination treatment experiments. Within these combinations, we analyze the most effective and synergistic drug pairs across all tested cell lines, considering the variations among cancers originating from different tissues. Our analysis reveals inhibitors of five DDR-related pathways (DNA topoisomerase, PLK1 kinase, p53-inducible ribonucleotide reductase, PARP, and cell cycle checkpoint proteins) that exhibit strong combinatorial efficacy and synergy when used alongside ATM/ATR/DNA-PK inhibitors. DNA damage is a key component of many cancer therapies. Here, the authors utilised a dose-response combination screen of ATR, ATM and DNA-PK inhibitors, and identified combination treatments that achieves high combinatorial efficacy and synergy. [ABSTRACT FROM AUTHOR]

Cancer is a genetic disease requiring multiple mutations for its development. However, many carcinogens are DNAunreactive and nonmutagenic and consequently described as nongenotoxic. One of such carcinogens is nickel, a global environmental pollutant abundantly emitted by burning of coal. We investigated activation of DNA damage responses by Ni and identified this metal as a replication stressor. Genotoxic stress markers indicated the accumulation of ssDNA and stalled replication forks, and Ni-treated cells were dependent on ATR for suppression of DNA damage and long-term survival. Replication stress by Ni resulted from destabilization of RRM1 and RRM2 subunits of ribonucleotide reductase and the resulting deficiency in dNTPs. Ni also increased DNA incorporation of rNMPs (detected by a specific fluorescent assay) and strongly enhanced their genotoxicity as a result of repressed repair of TOP1-DNA protein crosslinks (TOP1-DPC). The DPC-trap assay found severely impaired SUMOylation and K48-polyubiquitination of DNA-crosslinked TOP1 due to downregulation of specific enzymes. Our findings identified Ni as the human carcinogen inducing genome instability via DNA-embedded ribonucleotides and accumulation of TOP1-DPC which are carcinogenic abnormalities with poor detectability by the standard mutagenicity tests. The discovered mechanisms for Ni could also play a role in genotoxicity of other protein-reactive carcinogens. [ABSTRACT FROM AUTHOR]

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, the rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples from eight drilling sites with distinct geochemical and thermal profiles were selected for DNA extraction and PCR amplification to explore the diversity of methane-cycling archaea in the Guaymas Basin subsurface. We performed PCR amplifications with general (mcrIRD), and ANME-1 specific primers that target the alpha (α) subunit of methyl coenzyme M reductase (mcrA). Diverse ANME-1 lineages associated with anaerobic methane oxidation were detected in seven out of the eight drilling sites, preferentially around the methane-sulfate interface, and in several cases, showed preferences for specific sampling sites. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to marine mud volcanoes, seep sites, and the shallow marine subsurface. The most frequently recovered methanogenic phylotypes were closely affiliated with the hyperthermophilic Methanocaldococcaceae, and found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity in methanogen lineages. Our survey indicates the potential for extensive microbial methane cycling within subsurface sediments of Guaymas Basin. [ABSTRACT FROM AUTHOR]

Tumor cells generally require large amounts of nucleotides, and thus activate de novo purine synthesis (dnPS). In the dnPS reactions, 10-formyltetrahydorofolate (10-fTHF) supplied by one-carbon metabolism is utilized as a formyl group donor. We focused on aldehyde dehydrogenase 1 family member L1 (ALDH1L1), which metabolizes 10-fTHF to tetrahydrofolate and whose expression is often attenuated in hepatocellular carcinoma (HCC). We generated ALDH1L1-expressing HuH-7 cells to perform metabolome analysis and found that intracellular levels of serine were reduced and glycine was increased. In addition, 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), a dnPS intermediate, accumulated due to the consumption of 10-fTHF by ALDH1L1, which inhibited ZMP formylation. Importantly, ALDH1L1-expressing cells showed reduced ZMP sensitivity and higher mitochondrial activity. The suppression of mitochondrial serine catabolism by ALDH1L1 expression was speculated to be closely related to this phenotype. Gene set enrichment analysis utilizing The Cancer Genome Atlas data revealed that genes related to oxidative phosphorylation were enriched in HCC patients with high ALDH1L1 expression. Moreover, drug sensitivity data analysis demonstrated that HCC cell lines with low expression of ALDH1L1 were sensitive to ZMP and cordycepin, a structural analog of ZMP and AMP. Our study revealed that ZMP and AMP analogs might be effective in the pharmacotherapy of HCC patients with low expression of ALDH1L1. [ABSTRACT FROM AUTHOR]

The impact of N221S mutation in hRRM2B gene, which encodes the small subunit of human ribonucleotide reductase (RNR), on RNR activity and the pathogenesis of mitochondrial DNA depletion syndrome (MDDS) was investigated. Our results demonstrate that N221 mutations significantly reduce RNR activity, suggesting its role in the development of MDDS. We proposed an allosteric regulation pathway involving a chain of three phenylalanine residues on the αE helix of RNR small subunit β. This pathway connects the C-terminal loop of β2, transfers the activation signal from the large catalytic subunit α to β active site, and controls access of oxygen for radical generation. N221 is near this pathway and likely plays a role in regulating RNR activity. Mutagenesis studies on residues involved in the phenylalanine chain and the regulation pathway were conducted to confirm our proposed mechanism. We also performed molecular dynamic simulation and protein contact network analysis to support our findings. This study sheds new light on RNR small subunit regulation and provides insight on the pathogenesis of MDDS. [ABSTRACT FROM AUTHOR]

Proline is one of the proteinogenic amino acids. It is found in all kingdoms of life. It also has remarkable activity as an organocatalyst and is of structural importance in many folded polypeptides. Here, we show that prolinyl nucleotides with a phosphoramidate linkage are active building blocks in enzyme‐ and ribozyme‐free copying of RNA in the presence of monosubstituted imidazoles as organocatalysts. Both dinucleotides and mononucleotides are incorporated at the terminus of RNA primers in aqueous buffer, as instructed by the template sequence, in up to eight consecutive extension steps. Our results show that condensation products of amino acids and ribonucleotides can act like nucleoside triphosphates in media devoid of enzymes or ribozymes. Prolinyl nucleotides are metastable building blocks, readily activated by catalysts, helping to explain why the combination of α‐amino acids and nucleic acids was selected in molecular evolution. [ABSTRACT FROM AUTHOR]

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins. [ABSTRACT FROM AUTHOR]

Through infection and lysis of their coexisting bacterial hosts, viruses impact the biogeochemical cycles sustaining globally significant pelagic oceanic ecosystems. Currently, little is known of the ecological interactions between lytic viruses and their bacterial hosts underlying these biogeochemical impacts at ecosystem scales. This study focused on populations of lytic viruses carrying the B12-dependent Class II monomeric ribonucleotide reductase (RNR) gene, ribonucleotide-triphosphate reductase (Class II RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of Class II RTPR and the 16S rRNA gene, respectively. Amplicon sequence libraries were analyzed using compositional data analysis tools that account for the compositional nature of these data. Both virio- and bacterioplankton communities responded to environmental changes typically seen across seasonal cycles as well as shorter term upwelling–downwelling events. Defining Class II RTPR-carrying viral populations according to major phylogenetic clades proved a more robust means of exploring virioplankton ecology than operational taxonomic units defined by percent sequence homology. Virioplankton Class II RTPR populations showed positive associations with a broad phylogenetic diversity of bacterioplankton including dominant taxa within pelagic oceanic ecosystems such as Prochlorococcus and SAR11. Temporal changes in Class II RTPR virioplankton, occurring as both free viruses and within infected cells, indicated possible viral–host pairs undergoing sustained infection and lysis cycles throughout the seasonal study. Phylogenetic relationships inferred from Class II RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene. [ABSTRACT FROM AUTHOR]

The Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs) infect a wide range of eukaryotic species, including amoeba, algae, fish, amphibia, arthropods, birds, and mammals. This group of viruses has linear or circular double-stranded DNA genomes whose size spans approximately one order of magnitude, from 100 to 2500 kbp. The ultimate origin of this peculiar group of viruses remains an open issue. Some have argued that NCLDVs' origin may lie in a bacteriophage ancestor that increased its genome size by subsequent recruitment of eukaryotic and bacterial genes. Others have suggested that NCLDVs families originated from cells that underwent an irreversible process of genome reduction. However, the hypothesis that a number of NCLDVs sequences have been recruited from the host genomes has been largely ignored. In the present work, we have performed pangenomic analyses of each of the seven known NCLDVs families. We show that these families' core- and shell genes have cellular homologs, supporting possible escaping-gene events as part of its evolution. Furthermore, the detection of sequences that belong to two protein families (small chain ribonucleotide reductase and Erv1/Air) and to one superfamily [2OG-Fe(II) oxygenases] that are for distribution in all NCLDVs core and shell clusters encoding for oxygen-dependent enzymes suggests that the highly conserved core these viruses originated after the Proterozoic Great Oxidation Event that transformed the terrestrial atmosphere 2.4–2.3 Ga ago. [ABSTRACT FROM AUTHOR]

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that typically develop in the setting of neurofibromatosis type 1 (NF1) and cause significant morbidity. Conventional therapies are often ineffective for MPNSTs. Ribonucleotide reductase subunit M2 (RRM2) is involved in DNA synthesis and repair, and is overexpressed in multiple cancers. However, its role in NF1-associated MPNSTs remains unknown. Our objective was to determine the therapeutic and prognostic potential of RRM2 in NF1-associated MPNSTs. Methods: Identification of hub genes was performed by using NF1-associated MPNST microarray datasets. We detected RRM2 expression by immunochemical staining in an MPNST tissue microarray, and assessed the clinical and prognostic significance of RRM2 in an MPNST cohort. RRM2 knockdown and the RRM2 inhibitor Triapine were used to assess cell proliferation and apoptosis in NF1-associated MPNST cells in vitro and in vivo. The underlying mechanism of RRM2 in NF1-associated MPNST was revealed by transcriptome analysis. Results: RRM2 is a key hub gene and its expression is significantly elevated in NF1-associated MPNST. We revealed that high RRM2 expression accounted for a larger proportion of NF1-associated MPNSTs and confirmed the correlation of high RRM2 expression with poor overall survival. Knockdown of RRM2 inhibited NF1-associated MPNST cell proliferation and promoted apoptosis and S-phase arrest. The RRM2 inhibitor Triapine displayed dose-dependent inhibitory effects in vitro and induced significant tumor growth reduction in vivo in NF1-associated MPNST. Analysis of transcriptomic changes induced by RRM2 knockdown revealed suppression of the AKT-mTOR signaling pathway. Overexpression of RRM2 activates the AKT pathway to promote NF1-associated MPNST cell proliferation. Conclusions: RRM2 expression is significantly elevated in NF1-associated MPNST and that high RRM2 expression correlates with poorer outcomes. RRM2 acts as an integral part in the promotion of NF1-associated MPNST cell proliferation via the AKT-mTOR signaling pathway. Inhibition of RRM2 may be a promising therapeutic strategy for NF1-associated MPNST. [ABSTRACT FROM AUTHOR]

It is known that ribonucleotide reductase M2 (RRM2) could be induced by hepatitis B virus (HBV) via DNA damage response. However, whether RRM2 is a potential biomarker for diagnosing and monitoring liver fibrosis in chronic hepatitis B (CHB) patients is still unclear. In this study, CHB patients from GSE84044 (a transcriptome data from GEO data set) were downloaded and RRM2 was selected as a hub gene. Interestingly, a positive correlation was found between serum RRM2 and liver fibrosis stage. The similar results were found in CHB patients with normal alanine aminotransferase (ALT). Notably, RRM2 could effectively differentiate preliminary fibrosis from advanced fibrosis in CHB patients with/without normal ALT. In addition, RRM2 had a better performance in diagnosing liver fibrosis than two commonly used noninvasive methods (aspartate aminotransferase‐to‐platelet ratio index and fibrosis index based on the four factors), two classic fibrotic biomarkers (hyaluronic acid and type IV collagen) as well as Mac‐2 binding protein glycosylation isomer, a known serum fibrosis marker. Moreover, CHB patients with high RRM2, who were associated with advanced fibrosis, had higher expressions of immune checkpoints. Overall, serum RRM2 may be a promising biomarker for diagnosing and monitoring liver fibrosis in CHB patients. [ABSTRACT FROM AUTHOR]

Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression. Synopsis: The source of rNTPs used in mitochondrial transcription, which supports many key processes, is not well understood. This study shows that they are supplied through the phosphorylation of pyrimidine nucleosides via mitochondrial nucleoside diphosphate kinase NME6. NME6 sustains mitochondrial supply of pyrimidine nucleotides.NME6 ensures accumulation of mitochondrial transcripts and respiratory complexes.Pyrimidine nucleoside supplementation restores levels of mitochondrial transcripts and respiratory complexes in cells lacking NME6.NME6‐dependent nucleotide salvage maintains mtDNA when the cytosolic supply of pyrimidine deoxyribonucleotides is limited. [ABSTRACT FROM AUTHOR]

Objectives: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide. Most cases are diagnosed at an advanced stage using current tumor markers. Here, we aimed to identify potential novel potential biomarkers for NSCLC. Material/Methods: Four independent datasets from the Gene Expression Omnibus database were analyzed. The relative expression of ribonucleotide reductase regulatory subunit M2 (RRM2) mRNA in 30 paired of NSCLC paired tissues was measured by reverse transcription quantitative PCR. Serum levels of cytokeratin fragment 21–1 (CYFRA21-1), pro-gastrin-releasing peptide (ProGRP), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE) were measured using electrochemiluminescence immunoassays, and serum RRM2 levels were evaluated by an enzyme-linked immunosorbent assay. Results: The mRNA expression level of RRM2 was significantly increased in most NSCLC lesions compared to para-adjacent tissues. Serum RRM2 levels in NSCLC patients were significantly elevated compared to healthy controls and were also associated with distant metastasis and histological type, but not with tumor size or lymph node metastasis. Receiver operating characteristic curve analysis showed a higher diagnostic ratio for NSCLC using RRM2 alone compared to other traditional tumor markers. Conclusions: RRM2 is a potential sero-diagnostic biomarker for NSCLC. [ABSTRACT FROM AUTHOR]