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Journal of Experimental Botany Aug 2023The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal...
The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report a maize lost subsidiary cells (lsc) mutant, with many stomata lacking one or two SCs. The loss of SCs is supposed to have resulted from impeded subsidiary mother cell (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and striped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the concentration of dNTPs and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increased dNTP synthesis and promoted plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and growth of maize.
Topics: Zea mays; Ribonucleotide Reductases; Plant Stomata; Poaceae; Cell Differentiation; Arabidopsis
PubMed: 37103989
DOI: 10.1093/jxb/erad153 -
The ISME Journal Jul 2023Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable...
Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable phages (TBPs) are exceptional in terms of their unique lifestyle, especially their replicative transposition. Although several TBPs have been isolated and the life cycle of the representative phage Mu has been extensively studied, the diversity distribution and ecological functions of TBPs on the global scale remain unknown. Here, by mining TBPs from enormous microbial genomes and viromes, we established a TBP genome dataset (TBPGD), that expands the number of accessible TBP genomes 384-fold. TBPs are prevalent in diverse biomes and show great genetic diversity. Based on taxonomic evaluations, we propose the categorization of TBPs into four viral groups, including 11 candidate subfamilies. TBPs infect multiple bacterial phyla, and seem to infect a wider range of hosts than non-TBPs. Diverse auxiliary metabolic genes (AMGs) are identified in the TBP genomes, and genes related to glycoside hydrolases and pyrimidine deoxyribonucleotide biosynthesis are highly enriched. Finally, the influences of TBPs on their hosts are experimentally examined by using the marine bacterium Shewanella psychrophila WP2 and its infecting transposable phage SP2. Collectively, our findings greatly expand the genetic diversity of TBPs, and comprehensively reveal their potential influences in various ecosystems.
Topics: Bacteriophages; Ecosystem; Genome, Viral; Viruses; Bacteria
PubMed: 37069234
DOI: 10.1038/s41396-023-01414-z -
ELife Apr 2023Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and...
Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in and . deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both and is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction.
Topics: Ribonucleotides; Escherichia coli; Symbiosis; Ribonucleotide Reductases; Deoxyribonucleotides; Deoxyribonucleosides
PubMed: 37022136
DOI: 10.7554/eLife.83845 -
Cancer Chemotherapy and Pharmacology May 2023Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other...
INTRODUCTION
Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU.
METHODS
Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage.
RESULTS
We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU.
CONCLUSION
Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.
Topics: Humans; Thymidylate Synthase; Fluorodeoxyuridylate; Fluorouracil; Enzyme Inhibitors; Antimetabolites; Colorectal Neoplasms; DNA
PubMed: 37000221
DOI: 10.1007/s00280-023-04528-5 -
Frontiers in Endocrinology 2023Diabetes mellitus (DM) is on the rise, necessitating the development of novel therapeutic and preventive strategies to mitigate the disease's debilitating effects.... (Review)
Review
Diabetes mellitus (DM) is on the rise, necessitating the development of novel therapeutic and preventive strategies to mitigate the disease's debilitating effects. Diabetic cardiomyopathy (DCMP) is among the leading causes of morbidity and mortality in diabetic patients globally. DCMP manifests as cardiomyocyte hypertrophy, apoptosis, and myocardial interstitial fibrosis before progressing to heart failure. Evidence suggests that non-coding RNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), regulate diabetic cardiomyopathy-related processes such as insulin resistance, cardiomyocyte apoptosis and inflammation, emphasizing their heart-protective effects. This paper reviewed the literature data from animal and human studies on the non-trivial roles of miRNAs and lncRNAs in the context of DCMP in diabetes and demonstrated their future potential in DCMP treatment in diabetic patients.
Topics: Animals; Humans; MicroRNAs; Diabetic Cardiomyopathies; RNA, Long Noncoding; Deoxycytidine Monophosphate; Myocardium; Fibrosis; Diabetes Mellitus
PubMed: 36950696
DOI: 10.3389/fendo.2023.1124613 -
Bioconjugate Chemistry Mar 2023Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and...
Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.
PubMed: 36888923
DOI: 10.1021/acs.bioconjchem.2c00475 -
Critical Care (London, England) Mar 2023Community-acquired pneumonia (CAP) is the primary cause of death for children under five years of age globally. Hence, it is essential to investigate new early...
OBJECTIVE
Community-acquired pneumonia (CAP) is the primary cause of death for children under five years of age globally. Hence, it is essential to investigate new early biomarkers and potential mechanisms involved in disease severity.
METHODS
Proteomics combined with metabolomics was performed to identify biomarkers suitable for early diagnosis of severe CAP. In the training cohort, proteomics and metabolomics were performed on serum samples obtained from 20 severe CAPs (S-CAPs), 15 non-severe CAPs (NS-CAPs) and 15 healthy controls (CONs). In the verification cohort, selected biomarkers and their combinations were validated using ELISA and metabolomics in an independent cohort of 129 subjects. Finally, a combined proteomics and metabolomics analysis was performed to understand the major pathological features and reasons for severity of CAP.
RESULTS
The proteomic and metabolic signature was markedly different between S-CAPs, NS-CAPs and CONs. A new serum biomarker panel including 2 proteins [C-reactive protein (CRP), lipopolysaccharide (LBP)] and 3 metabolites [Fasciculol C, PE (14:0/16:1(19Z)), PS (20:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z))] was developed to identify CAP and to distinguish severe pneumonia. Pathway analysis of changes revealed activation of the cell death pathway, a dysregulated complement system, coagulation cascade and platelet function, and the inflammatory responses as contributors to tissue damage in children with CAP. Additionally, activation of glycolysis and higher levels of nucleotides led to imbalanced deoxyribonucleotide pools contributing to the development of severe CAP. Finally, dysregulated lipid metabolism was also identified as a potential pathological mechanism for severe progression of CAP.
CONCLUSION
The integrated analysis of the proteome and metabolome might open up new ways in diagnosing and uncovering the complexity of severity of CAP.
Topics: Child; Child, Preschool; Humans; Blood Coagulation; C-Reactive Protein; Cell Death; Community-Acquired Infections; Metabolomics; Proteomics; Pneumonia
PubMed: 36859478
DOI: 10.1186/s13054-023-04378-w -
International Journal of Medical... Mar 2023Staphylococcus aureus (S. aureus) is one of the critical clinical pathogens which can cause multiple diseases ranging from skin infections to fatal sepsis. S. aureus is... (Review)
Review
Staphylococcus aureus (S. aureus) is one of the critical clinical pathogens which can cause multiple diseases ranging from skin infections to fatal sepsis. S. aureus is generally considered to be an extracellular pathogen. However, more and more evidence has shown that S. aureus can survive inside various cells. Folate plays an essential role in multiple life activities, including the conversion of serine and glycine, the remethylation of homocysteine to methionine, and the de novo synthesis of purine /dTMP, et al. More and more studies reported that S. aureus intracellular infection requires the involvement of folate metabolism. This review focused on the mechanisms of folate metabolism and related substances affecting S. aureus infection. Loss of tetrahydrofolic acid (THF)-dependent dTMP directly inhibits the nucleotide synthesis pathway of the S. aureus due to pabA deficiency. Besides, trimethoprim-sulfamethoxazole (TMP/SMX), a potent antibiotic that treats S. aureus infections, interferes in the process of the folate mechanism and leads to the production of thymidine-dependent small-colony variants (TD-SCVs). In addition, S. aureus is resistant to lysostaphin in the presence of serine hydroxymethyltransferase (SHMT). We provide new insights for understanding the molecular pathogenesis of S. aureus infection.
Topics: Humans; Staphylococcus aureus; Thymidine Monophosphate; Staphylococcal Infections; Trimethoprim, Sulfamethoxazole Drug Combination; Folic Acid
PubMed: 36841056
DOI: 10.1016/j.ijmm.2023.151577 -
Clinical Hematology International Jun 2023Ribonucleotide Reductase (RNR) converts ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of subunits M1 and M2. It has been...
Ribonucleotide Reductase (RNR) converts ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of subunits M1 and M2. It has been studied as a prognostic factor in several solid tumors and in chronic hematological malignancies, but not in chronic lymphocytic leukemia (CLL). Peripheral blood samples were collected from 135 CLL patients. M1/M2 gene mRNA levels were measured and expressed as a RRM1-2/GAPDH ratio. M1 gene promoter methylation was studied in a patients' subgroup. M1 mRNA expression was higher in patients without anemia (p = 0.026), without lymphadenopathy (p = 0.005) and 17p gene deletion (p = 0.031). Abnormal LDH (p = 0.022) and higher Rai stage (p = 0.019) were associated with lower M1 mRNA levels. Higher M2 mRNA levels were found in patients without lymphadenopathy (p = .048), Rai stage 0 (p = 0.025) and Trisomy 12 (p = 0.025). The correlation between RNR subunits and clinic-biological characteristics in CLL patients demonstrate RNR's potential role as a prognostic factor.
PubMed: 36811764
DOI: 10.1007/s44228-023-00033-x -
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