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Journal of Cellular Physiology Jul 2023Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA...
Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMT . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans.
Topics: Animals; Humans; Chromosome Aberrations; DNA; Drosophila; Glycine Hydroxymethyltransferase; Pyridoxal Phosphate; Thymidine Monophosphate; Vitamin B 6
PubMed: 37183313
DOI: 10.1002/jcp.31033 -
Nucleosides, Nucleotides & Nucleic Acids 2023To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the...
To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2'-βD-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N-methyladenine, N-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2'-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.
Topics: Deoxyribonucleosides; Oligodeoxyribonucleotides; Solvents; Sequence Analysis, DNA; Kinetics
PubMed: 37165577
DOI: 10.1080/15257770.2023.2209135 -
Frontiers in Microbiology 2023Rhizosphere microbiota play an important role in regulating soil physical and chemical properties and improving crop production performance. This study analyzed the...
Rhizosphere microbiota play an important role in regulating soil physical and chemical properties and improving crop production performance. This study analyzed the relationship between the diversity of rhizosphere microbiota and the yield and quality of flue-cured tobacco at different transplant times (D30 group, D60 group and D90 group) and in different regions [Linxiang Boshang (BS) and Linxiang ZhangDuo (ZD)] by high-throughput sequencing technology. The results showed that there were significant differences in the physicochemical properties and rhizosphere microbiota of flue-cured tobacco rhizosphere soil at different transplanting times, and that the relative abundance of Bacillus in the rhizosphere microbiota of the D60 group was significantly increased. RDA and Pearson correlation analysis showed that , and were significantly correlated with soil physical and chemical properties. PIGRUSt2 function prediction results showed that compared with the D30 group, the D60 group had significantly increased metabolic pathways such as the superpathway of pyrimidine deoxyribonucleoside salvage, allantoin degradation to glyoxylate III and pyrimidine deoxyribonucleotides biosynthesis III metabolic pathways. The D90 group had significantly increased metabolic pathways such as ubiquitol-8 biosynthesis (prokaryotic), ubiquitol-7 biosynthesis (prokaryotic) and ubiquitol-10 biosynthesis (prokaryotic) compared with the D60 group. In addition, the yield and quality of flue-cured tobacco in the BS region were significantly higher than those in the ZD region, and the relative abundance of Firmicutes and Bacillus in the rhizosphere microbiota of flue-cured tobacco in the BS region at the D60 transplant stage was significantly higher than that in the ZD region. In addition, the results of the hierarchical sample metabolic pathway abundance map showed that the PWY-6572 metabolic pathway was mainly realized by , and that the relative abundance of flue-cured tobacco rhizosphere microbiota () participating in PWY-6572 in the D60 transplant period in the BS region was significantly higher than that in the ZD region. In conclusion, different transplanting periods of flue-cured tobacco have important effects on soil physical and chemical properties and rhizosphere microbial communities. There were significant differences in the rhizosphere microbiota and function of flue-cured tobacco in different regions, which may affect the performance and quality of this type of tobacco.
PubMed: 37152740
DOI: 10.3389/fmicb.2023.1141720 -
Frontiers in Microbiology 2023Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides...
INTRODUCTION
Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection.
METHODS
Bioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity was also evaluated.
RESULTS
ORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the enzymatic activity of RR.
CONCLUSION
These results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses.
PubMed: 37143536
DOI: 10.3389/fmicb.2023.1154840 -
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 -
Current Drug Metabolism 2023Therapeutic antisense oligonucleotides (ASOs) represent a diverse array of chemically modified singlestranded deoxyribonucleotides that work complementarily to affect...
Therapeutic antisense oligonucleotides (ASOs) represent a diverse array of chemically modified singlestranded deoxyribonucleotides that work complementarily to affect their mRNA targets. They vastly differ from conventional small molecules. These newly developed therapeutic ASOs possess unique absorption, distribution, metabolism, and excretion (ADME) processes that ultimately determine their pharmacokinetic, efficacy and safety profiles. The ADME properties of ASOs and associated key factors have not been fully investigated. Therefore, thorough characterization and in-depth study of their ADME properties are critical to support drug discovery and development processes for safe and effective therapeutic ASOs. In this review, we discussed the main factors affecting the ADME characteristics of these novels and evolving therapies. The major changes to ASO backbone and sugar chemistry, conjugation approaches, sites and routes of administration, are the principal determinants of ADME and PK profiles that consequentially impact their efficacy and safety profiles. In addition, species difference and DDI considerations are important in understanding ADME profile and PK translatability but are less studied for ASOs. We, therefore, have summarized these aspects based on current knowledge and provided discussions in this review. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of ASO drugs and provide future perspectives and knowledge gap analysis.
PubMed: 37076460
DOI: 10.2174/1389200224666230418092626 -
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 -
Pain Practice : the Official Journal of... Jul 2023Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field...
BACKGROUND
Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field include the utilization of programs that multiplex various signals to target different neural structures in the dorsal spinal cord associated with the painful area. Preclinical studies have been fundamental in understanding the mechanism by which this differential target multiplexed programming (DTMP) SCS approach works. Transcriptomic- and proteomic-based studies demonstrated that DTMP can modulate expression levels of genes and proteins involved in pain-related processes that have been affected by a neuropathic pain model. This work studied the effect of the intensity of DTMP signals on mechanical hypersensitivity and cell-specific transcriptomes.
METHODS
The spared nerve injury model (SNI) of neuropathic pain was induced in 20 animals which were 1:1 randomized into two SCS groups in which the intensity of the DTMP was adjusted to either 70% or 40% of the motor threshold (MT). SCS was applied continuously for 48 h via a quadripolar lead implanted in the dorsal epidural space of animals. Controls, which included a group of implanted SNI animals that received no SCS and a group of animals naive to the SNI, were assessed in parallel to the SCS groups. Mechanical hypersensitivity was assessed before SNI, before SCS, and at 48 h of SCS. At the end of SCS, the stimulated segment of the dorsal spinal cord was dissected and subjected to RNA sequencing to quantify expression levels in all experimental groups. Differential effects were assessed via fold-change comparisons of SCS and naive groups versus the no-SCS group for transcriptomes specific to neurons and glial cells. Standard statistical analyses were employed to assess significance of the comparisons (p < 0.05).
RESULTS
SCS treatments provided significant improvement in mechanical sensitivity relative to no SCS treatment. However, the change in the intensity did not provide a significant difference in the improvement of mechanical sensitivity. DTMP regulated expression levels back toward those found in the naive group in the cell-specific transcriptomes analyzed. There were no significant differences related to the intensity of the stimulation in terms of the percentage of genes in each transcriptome in which expression levels were reversed toward the naive state.
CONCLUSIONS
DTMP when applied at either 40% MT or 70% MT provided similar reduction of pain-like behavior in rats and similar effects in neuron- and glia-specific transcriptomes.
Topics: Rats; Animals; Pain Threshold; Pain Measurement; Proteomics; Thymidine Monophosphate; Disease Models, Animal; Neuralgia; Spinal Cord; Spinal Cord Stimulation
PubMed: 37067033
DOI: 10.1111/papr.13235 -
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 -
Chemical Research in Toxicology Apr 2023Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a...
Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant () in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.
Topics: Humans; Antineoplastic Agents; Doxorubicin; Deoxyguanine Nucleotides; Guanosine Triphosphate; Adenosine Triphosphate
PubMed: 37000908
DOI: 10.1021/acs.chemrestox.2c00367