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MSystems Apr 2023Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are...
Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an HO addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an HO addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.
Topics: Pseudomonas aeruginosa; Hydrogen Peroxide; Oxidative Stress; Reactive Oxygen Species; Deoxyribonucleotides
PubMed: 36794960
DOI: 10.1128/msystems.01005-22 -
International Journal of Molecular... Jan 2023Hepatitis C virus (HCV) frequently causes chronic infection in the human liver, which may progress to advanced hepatic fibrosis, cirrhosis, and hepatocellular carcinoma.... (Review)
Review
Hepatitis C virus (HCV) frequently causes chronic infection in the human liver, which may progress to advanced hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. HCV primarily infects highly differentiated quiescent hepatocytes and can modulate cell cycle-regulatory genes and proliferation pathways, which ultimately contribute to persistent infection and pathogenesis. On the other hand, several studies have shown differential regulation of HCV RNA and viral protein expression levels, depending on the proliferation state of hepatocytes and the phase of the cell cycle. HCV typically requires factors provided by host cells for efficient and persistent viral replication. Previously, we found that HCV infection upregulates the expression of ribonucleotide reductase subunit M2 (RRM2) in quiescent hepatocytes. RRM2 is a rate-limiting protein that catalyzes de novo synthesis of deoxyribonucleotide triphosphates, and its expression is highly regulated during various phases of the cell cycle. RRM2 functions as a pro-viral factor essential for HCV RNA synthesis, but its functional role in HCV-induced liver diseases remains unknown. Here, we present a comprehensive review of the role of the hepatocyte cell cycle, in correlation with RRM2 expression, in the regulation of HCV replication. We also discuss the potential relevance of this protein in the pathogenesis of HCV, particularly in the development of hepatocellular carcinoma.
Topics: Humans; Hepacivirus; Carcinoma, Hepatocellular; Hepatitis C; Hepatocytes; Liver Neoplasms; Virus Replication; Liver Cirrhosis; RNA
PubMed: 36768940
DOI: 10.3390/ijms24032619 -
Biomedicine & Pharmacotherapy =... Apr 2023Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in...
BACKGROUND
Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na] in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na].
METHODS
Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCX inhibitor. Myocardial [Na] was measured by Na NMR spectroscopy.
RESULTS
HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na] was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na] to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts.
CONCLUSIONS
Elevated myocardial [Na] contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na] and/or NCX may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na].
Topics: Mice; Male; Animals; Diabetic Cardiomyopathies; Sodium-Glucose Transporter 2 Inhibitors; Sodium; Calcium; Deoxycytidine Monophosphate; Myocardial Contraction; Mice, Inbred C57BL; Myocardium; Adenosine Triphosphate; Diabetes Mellitus
PubMed: 36731341
DOI: 10.1016/j.biopha.2023.114310 -
ACS Applied Materials & Interfaces Feb 2023The carbonyl groups of deoxyribonucleotide can resonantly couple with 53 THz middle infrared, which can highly transmit water without ionization-based damage to DNA...
The carbonyl groups of deoxyribonucleotide can resonantly couple with 53 THz middle infrared, which can highly transmit water without ionization-based damage to DNA molecules. Herein, we predict that vibrational coupling with THz irradiation could lower down the hybridization landscape of nucleic acids and thus affect DNA replication. Using polymerase chain reaction (PCR) as a measure, we found that THz shining can reduce the denature temperature of DNA duplexes by about 3 °C, which allows one to conduct PCR at lower temperature, facilitating long-time amplification reaction without losing enzymatic fidelity, i.e., normal PCR should be carried out at denaturing temperature ∼4 °C higher than the melting temperature (), but THz-PCR only requires temperature ∼1 °C higher than due to the nonthermal effect of THz shining. Moreover, the melting time can also be shortened to 1/5 due to the enhanced vibration coupling with 53 THz irradiation. We proposed THz-PCR as an innovated DNA amplification technique with ultrahigh specificity and sensitivity and also successfully demonstrated its advantages in forensic detections.
Topics: Vibration; DNA; Polymerase Chain Reaction; Temperature; Nucleic Acid Amplification Techniques
PubMed: 36724344
DOI: 10.1021/acsami.2c22413 -
Free Radical Biology & Medicine Feb 2023Oxidative stress can attack precursor nucleotides, resulting in nucleic acid damage in cells. It remains unclear how 8-oxo-dGTP and 8-oxoGTP, oxidized forms of dGTP and...
Oxidative stress can attack precursor nucleotides, resulting in nucleic acid damage in cells. It remains unclear how 8-oxo-dGTP and 8-oxoGTP, oxidized forms of dGTP and GTP, respectively, could affect DNA or RNA oxidation levels and tumor development. To address this, we intravenously administered 8-oxo-dGTP and 8-oxoGTP to wild-type and MTH1-knockout mice. 8-oxoGTP administration increased frequency of tumor incidence, which is more prominent in MTH1-knockout mice. However, 8-oxo-dGTP treatment rather reduced tumor development regardless of the mouse genotype. The tumor suppressive effects of 8-oxo-dGTP were further confirmed using xenograft and C57/6J-Apc/Nju mouse models. Mechanistically, 8-oxo-dGTP increased the 8-oxo-dG contents in DNA and DNA strand breakage, induced cell cycle arrest in S phase and apoptosis mediated by AIF, eventually leading to reduced tumor incidence. These results suggest distinct roles of 8-oxo-dGTP and 8-oxoGTP in tumor development.
Topics: Humans; Animals; Mice; Phosphoric Monoester Hydrolases; S Phase; Deoxyguanine Nucleotides; Neoplasms; DNA; Mice, Knockout; Apoptosis; DNA Repair Enzymes
PubMed: 36640852
DOI: 10.1016/j.freeradbiomed.2023.01.012 -
Nucleosides, Nucleotides & Nucleic Acids 2023We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl...
We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl phosphonate esters without 1'-α-anomerization, providing an improved synthesis of 5'-methylene substituted 2',5'-deoxynucleotides.
Topics: Esters; Organophosphonates; Deoxyribonucleotides; Demethylation
PubMed: 36629008
DOI: 10.1080/15257770.2023.2166064 -
Medicine Dec 2022This prospective cohort study explored whether body constitution (BC) independently predicts new-onset albuminuria in persons with type 2 diabetes mellitus (T2DM)...
Traditional Chinese medicine body constitution predicts new-onset diabetic albuminuria in patients with type 2 diabetes: Taichung diabetic body constitution prospective cohort study.
This prospective cohort study explored whether body constitution (BC) independently predicts new-onset albuminuria in persons with type 2 diabetes mellitus (T2DM) enrolled in the diabetes care management program (DCMP) of a medical center, providing evidence of integrating traditional Chinese medicine into DCMP for improving care quality. Persons with T2DM (n = 426) originally without albuminuria enrolled in DCMP were recruited in 2010 and were then followed up to 2015 for detecting new-onset albuminuria. The participants received urinalysis and blood test annually. Albuminuria was determined by an elevated urinary albumin/creatinine ratio (≥ 30 µg/mg), and poor glucose control was defined as Glycosylated hemoglobin above or equal to 7%. BC type (Yin deficiency, Yang deficiency, and phlegm stasis) was assessed using a well-validated body constitution questionnaire at baseline. Risk factors for albuminuria (sociodemographic factors, diabetes history, lifestyle behaviors, lipid profile, blood pressure, and kidney function) were also recorded. Hazard ratios (HR) of albuminuria for BC were estimated using multivariate Cox proportional hazards model. During the 4-year follow-up period, albuminuria occurred in 30.5% of participants (n = 130). The HR indicated that Yin deficiency was significantly associated with an increased risk of new-onset albuminuria in persons with T2DM and good glucose control after adjustment for other risk factors (HR = 2.09; 95% confidence interval = 1.05-4.17, P = .04), but not in those with poor glucose control. In persons with T2DM and poor glucose control, phlegm stasis was also significantly associated with a higher risk of albuminuria (2.26; 1.03-4.94, P = .04) after multivariate adjustment, but not in those with good glucose control. In addition to already-known risk factors, BC is an independent and significant factor associated with new-onset albuminuria in persons with T2DM. Our results imply Yin deficiency and phlegm stasis interacting with glucose control status may affect new-onset albuminuria in persons with T2DM.
Topics: Humans; Albuminuria; Blood Glucose; Body Constitution; Deoxycytidine Monophosphate; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Medicine, Chinese Traditional; Prospective Studies; Yin Deficiency
PubMed: 36550881
DOI: 10.1097/MD.0000000000032342 -
ELife Dec 2022Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting...
Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions.
Topics: Protein Serine-Threonine Kinases; Cell Cycle Proteins; Saccharomyces cerevisiae Proteins; Checkpoint Kinase 2; DNA Replication; Saccharomyces cerevisiae; Cell Cycle; Cell Cycle Checkpoints; DNA Damage; Phosphorylation
PubMed: 36520028
DOI: 10.7554/eLife.84320 -
Accounts of Chemical Research Dec 2022The development of the liquid microjet technique by Faubel and co-workers has enabled the investigation of high vapor pressure liquids and solutions utilizing...
The development of the liquid microjet technique by Faubel and co-workers has enabled the investigation of high vapor pressure liquids and solutions utilizing high-vacuum methods. One such method is photoelectron spectroscopy (PES), which allows one to probe the electronic properties of a sample through ionization in a state-specific manner. Liquid microjets consisting of pure solvents and solute-solvent systems have been studied with great success utilizing PES and, more recently, time-resolved PES (TRPES). Here, we discuss progress made over recent years in understanding the solvation and excited state dynamics of the solvated electron and nucleic acid constituents (NACs) using these methods, as well as the prospect for their future.The solvated electron is of particular interest in liquid microjet experiments as it represents the simplest solute system. Despite this simplicity, there were still many unresolved questions about its binding energy and excited state relaxation dynamics that are ideal problems for liquid microjet PES. In the work discussed in this Account, accurate binding energies were measured for the solvated electron in multiple high vapor pressure solvents. The advantages of liquid jet PES were further highlighted in the femtosecond excited state relaxation studies on the solvated electron in water where a 75 ± 20 fs lifetime attributable to internal conversion from the excited p-state to a hot ground state was measured, supporting a nonadiabatic relaxation mechanism.Nucleic acid constituents represent a class of important solutes with several unresolved questions that the liquid microjet PES method is uniquely suited to address. As TRPES is capable of tracking dynamics with state-specificity, it is ideal for instances where there are multiple excited states potentially involved in the dynamics. Time-resolved studies of NAC relaxation after excitation using ultraviolet light identified relaxation lifetimes from multiple excited states. The state-specific nature of the TRPES method allowed us to identify the lack of any signal attributable to the nπ* state in thymine derived NACs. The femtosecond time resolution of the technique also aided in identifying differences between the excited state lifetimes of thymidine and thymidine monophosphate. These have been interpreted, aided by molecular dynamics simulations, as an influence of conformational differences leading to a longer excited state lifetime in thymidine monophosphate.Finally, we discuss advances in tabletop light sources extending into the extreme ultraviolet and soft X-ray regimes that allow expansion of liquid jet TRPES to full valence band and potentially core level studies of solutes and pure liquids in liquid microjets. As most solutes have ground state binding energies in the range of 10 eV, observation of both excited state decay and ground state recovery using ultraviolet pump-ultraviolet probe TRPES has been intractable. With high-harmonic generation light sources, it will be possible to not only observe complete relaxation pathways for valence level dynamics but to also track dynamics with element specificity by probing core levels of the solute of interest.
Topics: Humans; Photoelectron Spectroscopy; Thymidine Monophosphate; Solvents; Water; Molecular Dynamics Simulation
PubMed: 36480155
DOI: 10.1021/acs.accounts.2c00609 -
Langmuir : the ACS Journal of Surfaces... Dec 2022The fate of biomolecules in the environment depends in part on understanding the surface chemistry occurring at the biological-geochemical (bio-geo) interface. Little is...
The fate of biomolecules in the environment depends in part on understanding the surface chemistry occurring at the biological-geochemical (bio-geo) interface. Little is known about how environmental DNA (eDNA) or smaller components, like nucleotides and oligonucleotides, persist in aquatic environments and the role of surface interactions. This study aims to probe surface interactions and adsorption behavior of nucleotides on oxide surfaces. We have investigated the interactions of individual nucleotides (dGMP, dCMP, dAMP, and dTMP) on TiO particle surfaces as a function of pH and in the presence of complementary and noncomplementary base pairs. Using attenuated total reflectance-Fourier transform infrared spectroscopy, there is an increased number of adsorbed nucleotides at lower pH with a preferential interaction of the phosphate group with the oxide surface. Additionally, differential adsorption behavior is seen where purine nucleotides are preferentially adsorbed, with higher surface saturation coverage, over their pyrimidine derivatives. These differences may be a result of intermolecular interactions between coadsorbed nucleotides. When the TiO surface was exposed to two-component solutions of nucleotides, there was preferential adsorption of dGMP compared to dCMP and dTMP, and dAMP compared to dTMP and dCMP. Complementary nucleotide base pairs showed hydrogen-bond interactions between a strongly adsorbed purine nucleotide layer and a weaker interacting hydrogen-bonded pyrimidine second layer. Noncomplementary base pairs did not form a second layer. These results highlight several important findings: (i) there is differential adsorption of nucleotides; (ii) complementary coadsorbed nucleotides show base pairing with a second layer, and the stability depends on the strength of the hydrogen bonding interactions and; (iii) the first layer coverage strongly depends on pH. Overall, the importance of surface interactions in the adsorption of nucleotides and the templating of specific interactions between nucleotides are discussed.
Topics: Thymidine Monophosphate; Deoxycytidine Monophosphate; Oxides; Hydrogen Bonding; Hydrogen
PubMed: 36445255
DOI: 10.1021/acs.langmuir.2c01604