The skin, serving as the body\'s primary defense against external elements, plays a crucial role in protecting the body from infections and injuries, as well as maintaining overall homeostasis. Skin aging, a common manifestation of the aging process, involves the gradual deterioration of its normal structure and repair mechanisms. Addressing the issue of skin aging is increasingly imperative. Multiple pieces of evidence indicate the potential anti-aging effects of exogenous nucleotides (NTs) through their ability to inhibit oxidative stress and inflammation. This study aims to investigate whether exogenous NTs can slow down skin aging and elucidate the underlying mechanisms. To achieve this objective, senescence-accelerated mouse prone-8 (SAMP8) mice were utilized and randomly allocated into Aging, NTs-low, NTs-middle, and NTs-high groups, while senescence-accelerated mouse resistant 1 (SAMR1) mice were employed as the control group. After 9 months of NT intervention, dorsal skin samples were collected to analyze the pathology and assess the presence and expression of substances related to the aging process. The findings indicated that a high-dose NT treatment led to a significant increase in the thickness of the epithelium and dermal layers, as well as Hyp content (p < 0.05). Additionally, it was observed that low-dose NT intervention resulted in improved aging, as evidenced by a significant decrease in p16 expression (p < 0.05). Importantly, the administration of high doses of NTs could improve, in some ways, mitochondrial function, which is known to reduce oxidative stress and promote ATP and NAD+ production significantly. These observed effects may be linked to NT-induced autophagy, as evidenced by the decreased expression of p62 and increased expression of LC3BI/II in the intervention groups. Furthermore, NTs were found to upregulate pAMPK and PGC-1α expression while inhibiting the phosphorylation of p38MAPK, JNK, and ERK, suggesting that autophagy may be regulated through the AMPK and MAPK pathways. Therefore, the potential induction of autophagy by NTs may offer benefits in addressing skin aging through the activation of the AMPK pathway and the inhibition of the MAPK pathway.

Nucleotides (NTs) act as pivotal regulatory factors in numerous biological processes, playing indispensable roles in growth, development, and metabolism across organisms. This study delves into the effects of exogenous NTs on hepatic insulin resistance using palmitic-acid-induced HepG2 cells, administering interventions at three distinct dosage levels of exogenous NTs. The findings underscore that exogenous NT intervention augments glucose consumption in HepG2 cells, modulates the expression of glycogen-synthesis-related enzymes (glycogen synthase kinase 3β and glycogen synthase), and influences glycogen content. Additionally, it governs the expression levels of hepatic enzymes (hexokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase). Moreover, exogenous NT intervention orchestrates insulin signaling pathway (insulin receptor substrate-1, protein kinase B, and forkhead box protein O1) and AMP-activated protein kinase (AMPK) activity in HepG2 cells. Furthermore, exogenous NT intervention fine-tunes the expression levels of oxidative stress-related markers (malondialdehyde, glutathione peroxidase, and NADPH oxidase 4) and the expression of inflammation-related nuclear transcription factor (NF-κB). Lastly, exogenous NT intervention regulates the expression levels of glucose transporter proteins (GLUTs). Consequently, exogenous NTs ameliorate insulin resistance in HepG2 cells by modulating the IRS-1/AKT/FOXO1 pathways and regulate glucose consumption, glycogen content, insulin signaling pathways, AMPK activity, oxidative stress, and inflammatory status.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA\'s association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

C2\'-halogenation has been recognized as an essential modification to enhance the drug-like properties of nucleotide analogs. The direct C2\'-halogenation of the nucleotide 2\'-deoxyadenosine-5\'-monophosphate (dAMP) has recently been achieved using the Fe(II)/α-ketoglutarate-dependent nucleotide halogenase AdaV. However, the limited substrate scope of this enzyme hampers its broader applications. In this study, we report two halogenases capable of halogenating 2\'-deoxyguanosine monophosphate (dGMP), thereby expanding the family of nucleotide halogenases. Computational studies reveal that nucleotide specificity is regulated by the binding pose of the phosphate group. Based on these findings, we successfully engineered the substrate specificity of these halogenases by mutating second-sphere residues. This work expands the toolbox of nucleotide halogenases and provides insights into the regulation mechanism of nucleotide specificity.

The DNA building blocks 2\'-deoxynucleotides are enantiomeric, with their natural β-D-configuration dictated by the sugar moiety. Their synthetic β-L-enantiomers (βLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual βLdNs embedded in D-DNA. Here, we address the template properties of βLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by βLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase β treated βLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process βLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize βLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine βLdNs were resistant to repair in human cells, whereas purine βLdNs appear to be partly repaired. Overall, βLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

The FDA has approved several drugs based on the fluorinated nucleoside pharmacophore, and numerous drugs are currently in clinical trials. Fluorine-containing nucleos(t)ides offer significant antiviral and anticancer activity. The insertion of a fluorine atom, either in the base or sugar of nucleos(t)ides, alters its electronic and steric parameters and transforms the lipophilicity, pharmacodynamic, and pharmacokinetic properties of these moieties. The fluorine atom restricts the oxidative metabolism of drugs and provides enzymatic metabolic stability towards the glycosidic bond of the nucleos(t)ide. The incorporation of fluorine also demonstrates additional hydrogen bonding interactions in receptors with enhanced biological profiles. The present article discusses the synthetic methodology and antiviral activities of FDA-approved drugs and ongoing fluoro-containing nucleos(t)ide drug candidates in clinical trials.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2\'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2\'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

Nucleotide-binding site (NBS) domain genes are one of the superfamily of resistance genes involved in plant responses to pathogens. The current study identified 12,820 NBS-domain-containing genes across 34 species covering from mosses to monocots and dicots. These identified genes are classified into 168 classes with several novel domain architecture patterns encompassing significant diversity among plant species. Several classical (NBS, NBS-LRR, TIR-NBS, TIR-NBS-LRR, etc.) and species-specific structural patterns (TIR-NBS-TIR-Cupin_1-Cupin_1, TIR-NBS-Prenyltransf, Sugar_tr-NBS etc.) were discovered. We observed 603 orthogroups (OGs) with some core (most common orthogroups; OG0, OG1, OG2, etc.) and unique (highly specific to species; OG80, OG82, etc.) OGs with tandem duplications. The expression profiling presented the putative upregulation of OG2, OG6, and OG15 in different tissues under various biotic and abiotic stresses in susceptible and tolerant plants to cotton leaf curl disease (CLCuD). The genetic variation between susceptible (Coker 312) and tolerant (Mac7) Gossypium hirsutum accessions identified several unique variants in NBS genes of Mac7 (6583 variants) and Coker312 (5173 variants). The protein-ligand and proteins-protein interaction showed a strong interaction of some putative NBS proteins with ADP/ATP and different core proteins of the cotton leaf curl disease virus. The silencing of GaNBS (OG2) in resistant cotton through virus-induced gene silencing (VIGS) demonstrated its putative role in virus tittering. The presented study will be further helpful in understanding the plant adaptation mechanism.

This study investigated the effects of supplemental nucleotides, autolyzed yeast (Saccharomyces cerevisiae), and sodium butyrate in diets for nursery pigs on growth performance, diarrhea incidence, blood profile, intestinal morphology, mRNA expression of nutrient transporters, inflammatory markers, antioxidant profile, and tight junction proteins in the small intestine. One hundred eighty 21-day-old pigs (5.17 ± 0.57 kg) were assigned in a randomized block design to 1 of 4 dietary treatments: (1) CON: control, basal diet, (2) NUC: CON + nucleotides, (3) YSC: CON + lysed yeast S. cerevisiae, (4) ASB: CON + acidifier sodium butyrate. Pigs were fed for 24 days, phase 1 (21-32 days) and 2 (32-45 days). During phase 1, YSC and ASB improved average daily gain (ADG) and feed conversion (FC) compared with CON. At the overall period, ASB improved ADG and YSC improved FC compared with CON. The NUC diet did not affect growth performance. The ASB increased ileal villus height compared to CON. The YSC and ASB reduced the number of Peyer\'s patches in the ileum compared with CON. The YSC increased mRNA expression of nutrient transporters (SMCT2, MCT1, and PepT1), tight junction proteins (OCL and ZO-1), antioxidants (GPX), and IL1-β in the jejunum compared with CON. The ASB increased mRNA expression of nutrient transporters (SGLT1 and MCT1), tight junction proteins (OCL and ZO-1), and antioxidants (GPX and SOD) compared with CON. In conclusion, autolyzed yeast and sodium butyrate promoted growth performance by improving the integrity of the intestinal barrier, the mRNA expression of nutrient transporters, and antioxidant enzymes in the jejunum of nursery pigs whereas supplementation of nucleotides did not show such effects.