Search Results (53)

Plant microRNAs (miRNAs) are endogenous non-coding RNAs (~20–24 nucleotides) that regulate gene expression post-transcriptionally, playing critical roles in plant growth, development, and stress responses. This review systematically examines AI applications in plant miRNA research, tracing evolution from traditional machine learning to deep learning architectures. Plant miRNAs exhibit distinctive features necessitating plant-specific computational approaches: nuclear-localized biogenesis, high target complementarity (>80%), and coding region targeting. These characteristics enable more accurate computational prediction and experimental validation than animal systems. Methodological advances have improved prediction accuracy from ~90% (early SVMs) to >99% (recent deep learning), though metrics reflect different evaluation contexts. We analyze applications across miRNA identification, target prediction with degradome validation, miRNA–lncRNA interactions, and ceRNA networks. Critical assessment reveals that degradome data capture mixed RNA fragments from multiple sources beyond miRNA cleavage, requiring stringent multi-evidence validation. Similarly, fundamental ambiguities in lncRNA definition compound prediction uncertainties. Major challenges include severe data imbalance (positive to negative ratios of 1:100 to 1:10,000), limited cross-species generalization, insufficient model interpretability, and experimental validation bottlenecks. Approximately 75% of plant miRNA families in miRBase v20 lack convincing evidence, underscoring the need for rigorous annotation standards. Future directions encompass multimodal deep learning, explainable AI, spatiotemporal graph neural networks, and ultimately AI-driven de novo miRNA design, though the latter requires substantial advances in both computation and high-throughput validation. This synthesis demonstrates that AI has become indispensable for plant miRNA research, providing essential support for crop improvement while acknowledging persistent challenges demanding continued innovation. Full article

Polynucleotide (PN) and polydeoxyribonucleotide (PDRN) are DNA-derived biopolymers increasingly recognized for their potential in dermatology. Despite their structural similarities, PN and PDRN exhibit distinct functions due to differences in polymer length and molecular weight. PN, composed of longer DNA fragments, plays a key role in extracellular matrix remodeling. Conversely, PDRN, composed of relatively shorter oligonucleotide sequences than those of PN, enhances skin condition through adenosine receptor activations and supports nucleotide synthesis via both the salvage and de novo pathways. This review provides a critical comparison of the molecular characteristics and functions of PN and PDRN with particular emphasis on their dermatological applications. By delineating their respective roles in esthetic and regenerative medicine, we aim to highlight recent advances that may guide the development of optimized treatment strategies and foster evidence-based clinical practice. Full article

Dihydroorotase (DHOase; EC 3.5.2.3) is a zinc-dependent metalloenzyme that plays a key role in the de novo pyrimidine biosynthesis pathway, catalyzing the reversible cyclization of N-carbamoyl aspartate to dihydroorotate. This reaction is essential for the production of uridine monophosphate, the precursor of all pyrimidine nucleotides required for DNA and RNA synthesis. Despite its conserved enzymatic function, DHOase exhibits significant structural diversity across species, particularly in its oligomeric states, gene fusion patterns, and active site architecture. A crucial structural feature of DHOase is its flexible active site loop, which undergoes dynamic conformational changes during catalysis. Previously, the loop-in conformation was associated with substrate binding, whereas the loop-out conformation was linked to product release and non-substrate ligand binding. However, recent crystallographic studies challenge this paradigm, revealing that certain non-substrate ligands and inhibitors, including malate, 5-fluoroorotate, plumbagin, 5-aminouracil, and 5-fluorouracil, interact with DHOase via a loop-in binding mechanism rather than the previously assumed loop-out mode. These findings necessitate a reassessment of the catalytic mechanism of DHOase and underscore the active site loop as a potential target for drug development. This review revisits the structural and biochemical mechanisms of DHOase, with a focus on recent crystallographic insights that redefine the loop-in binding mode for ligand interaction. By leveraging the unique conformational dynamics of the active site loop, novel inhibitors may be developed to selectively target pyrimidine biosynthesis in cancer cells and microbial pathogens. These insights emphasize the crucial role of structural biology in therapeutic design and highlight DHOase as a promising drug target. Full article

Every year, dengue virus affects hundreds of millions of individuals worldwide. To date, there is no specific medication to treat dengue virus infections. Nucleobases, the base of a nucleoside without ribose, are understudied as potential treatments for viral infections. Antiviral nucleobases are converted in infected cells to their corresponding nucleoside triphosphate active form. Importantly, the conversion of nucleobases to their active nucleotide form and their antiviral effect can be enhanced when combined with de novo nucleotide biosynthesis inhibitors. In this work, we evaluated seven purine and pyrimidine nucleobases alone or combined with six purine or pyrimidine de novo nucleotide biosynthesis inhibitors, including novel prodrugs. Our study revealed that while a strong potentiation of purine nucleobases by purine de novo nucleotide biosynthesis inhibitors was observed, the pyrimidine nucleobases were not potentiated by pyrimidine de novo nucleotide biosynthesis inhibitors, possibly highlighting a significant difference between the modulation of purine versus pyrimidine de novo pathways and their impact on nucleobase potentiation. Most significant antiviral effects and potentiation were observed for Favipiravir, T-1105, and ribavirin nucleobases combined with purine nucleotide de novo synthesis inhibitors. These results are significant because drug combinations may solve the limited efficacy observed for some antiviral nucleobase drugs such as Favipiravir. Full article

De novo synthesis of phage genomes enables flexible genome modification and simplification. This study explores the synthetic genome assembly of Pseudomonas phage vB_PaeS_SCUT-S4 (S4), a 42,932 bp headful packaging phage, which encapsidates a terminally redundant, double-stranded DNA genome exceeding unit length. We demonstrate that using the yeast TAR approach, the S4 genome can be assembled and rebooted from a unit-length genome plus a minimal 60 bp terminal redundant sequence. Furthermore, we show that S4 can be synthesized from arbitrary starting nucleotides and modified with a red fluorescent protein as a reporter. Additionally, we successfully designed and assembled synthetic S4 phages with reduced genomes, knocking out up to 10 of the 24 hypothetical genes simultaneously, with a combined length of 2883 bp, representing 6.7% of the unit-length genome. This work highlights the potential for engineering simplified, customizable headful packaging phage genomes, providing a foundation for future studies of these phages for potential clinical applications. Full article

The SCD is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids (MUFAs) in dairy cows; however, its role in the mammary gland of buffalo is not well understood. In this study, we isolated and characterized the complete coding sequence (CDS) of the buffalo SCD gene from mammary gland tissue and investigated its effects on milk fat synthesis using bioinformatics analyses, tissue differential expression detection, and cellular functional experiments. The cloned SCD gene has a CDS length of 1080 bp, encoding a protein of 359 amino acids. This protein is hydrophilic, lacks a signal peptide, and contains four transmembrane domains, including 10 conserved motifs and a Delta9-FADS domain, characteristic of the fatty acid desaturase family involved in unsaturated fatty acid biosynthesis within the endoplasmic reticulum. Molecular characterization revealed that the physicochemical properties, conserved domains, structures, and functions of buffalo SCD are highly similar to those in other Bovidae species. Among the tissues analyzed, SCD expression was highest in the mammary gland during lactation and in the cerebellum during dry-off period. Notably, SCD expression in the mammary gland was significantly higher during lactation compared to the dry-off period. Subcellular localization experiments confirmed that SCD functions in the endoplasmic reticulum of buffalo mammary epithelial cells (BuMECs). Functional overexpression and interference experiments in BuMECs demonstrated that SCD promotes milk fat synthesis by affecting the expression of lipid synthesis-related genes such as ACACA, FASN, and DGAT1, as well as milk fat regulatory genes like SREBFs and PPARG, thereby influencing intracellular triglyceride (TAG) content. Additionally, 18 single-nucleotide polymorphisms (SNPs) were identified in the buffalo SCD gene, with a specific SNP at c.-605, showing potential as molecular markers for improving milk production traits. These findings highlight that the SCD gene is a key gene in buffalo milk fat synthesis, involved in the de novo synthesis of milk fatty acids. Full article

Background: mTORC1 activity is dependent on the presence of micronutrients, including Asparagine (Asn), to promote anabolic cell signaling in many cancers. We hypothesized that targeting Asn metabolism would inhibit tumor growth by reducing mTORC1 activity in well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS). Methods: Human tumor metabolomic analysis was utilized to compare abundance of Asn in WD vs. DD LPS. Gene set enrichment analysis (GSEA) compared relative expression among metabolic pathways upregulated in DD vs. WD LPS. Proliferation assays were performed for LPS cell lines and organoid models by using the combination treatment of electron transport chain (ETC) inhibitors with Asn-free media. ¹³C-Glucose-labeling metabolomics evaluated the effects of combination treatment on nucleotide synthesis. Murine xenograft models were used to assess the effects of ETC inhibition combined with PEGylated L-Asparaginase (PEG-Asnase) on tumor growth and mTORC1 signaling. Results: Asn was enriched in DD LPS compared to WD LPS. GSEA indicated that mTORC1 signaling was upregulated in DD LPS. Within available LPS cell lines and organoid models, the combination of ETC inhibition with Asn-free media resulted in reduced cell proliferation. Combination treatment inhibited nucleotide synthesis and promoted cell cycle arrest. In vivo, the combination of ETC inhibition with PEG-Asnase restricted tumor growth. Conclusions: Asn enrichment and mTORC1 upregulation are important factors contributing to WD/DD LPS tumor progression. Effective targeting strategies require limiting access to extracellular Asn and inhibition of de novo synthesis mechanisms. The combination of PEG-Asnase with ETC inhibition is an effective therapy to restrict tumor growth in WD/DD LPS. Full article

Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased “de novo” synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the “de novo” synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways. Full article

Phytoplasmas are linked to diseases in hundreds of economically important crops, including carrots. In carrots, phytoplasmosis is associated with leaf chlorosis and necrosis, coupled with inhibited root system development, ultimately leading to significant economic losses. During a field study conducted in Baden-Württemberg (Germany), two strains of the provisional taxon ‘Candidatus Phytoplasma asteris’ were identified within a carrot plot. For further analysis, strains M8 and M33 underwent shotgun sequencing, utilising single-molecule-real-time (SMRT) long-read sequencing and sequencing-by-synthesis (SBS) paired-end short-read sequencing techniques. Hybrid assemblies resulted in complete de novo assemblies of two genomes harboring circular chromosomes and two plasmids. Analyses, including average nucleotide identity and sequence comparisons of established marker genes, confirmed the phylogenetic divergence of ‘Ca. P. asteris’ and a different assignment of strains to the 16S rRNA subgroup I-A for M33 and I-B for M8. These groups exhibited unique features, encompassing virulence factors and genes, associated with the mobilome. In contrast, pan-genome analysis revealed a highly conserved gene set related to metabolism across these strains. This analysis of the Aster Yellows (AY) group reaffirms the perception of phytoplasmas as bacteria that have undergone extensive genome reduction during their co-evolution with the host and an increase of genome size by mobilome. Full article

CTP synthase (CTPS), the rate-limiting enzyme in the de novo synthesis of CTP, assembles into a filamentous structure termed the cytoophidium. The Hippo pathway regulates cell proliferation and apoptosis. The relationship of the nucleotide metabolism with the Hippo pathway is little known. Here, we study the impact of the Hippo pathway on the cytoophidium in Drosophila melanogaster posterior follicle cells (PFCs). We find that the inactivation of the Hippo pathway correlates with reduced cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigates hpo (Hippo)-associated over-proliferation. In summary, our results suggest that there is a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs. Full article

Endometrial carcinoma (EC) is the most common gynecological malignancy in the United States. De novo pyrimidine synthesis pathways generate nucleotides that are required for DNA synthesis. Approximately 38% of human endometrial tumors present with an overexpression of human dihydroorotate dehydrogenase (DHODH). However, the role of DHODH in cancer cell DNA replication and its impact on modulating a treatment response is currently unknown. Here, we report that endometrial tumors with overexpression of DHODH are associated with a high mutation count and chromosomal instability. Furthermore, tumors with an overexpression of DHODH show significant co-occurrence with mutations in DNA replication polymerases, which result in a histologically high-grade endometrial tumor. An in vitro experiment demonstrated that the inhibition of DHODH in endometrial cancer cell lines significantly induced replication-associated DNA damage and hindered replication fork progression. Furthermore, endometrial cancer cells were sensitive to the DHODH inhibitor either alone or in combination with the Poly (ADP-ribose) polymerase 1 inhibitor. Our findings may have important clinical implications for utilizing DHODH as a potential target to enhance cytotoxicity in high-grade endometrial tumors. Full article

This study compared the growth performance, serum biochemical indicators, rumen fermentation parameters, rumen bacterial structure, and fecal bacterial structure of cattle and yaks fed for two months and given a feed containing concentrate of a roughage ratio of 7:3 on a dry matter basis. Compared with cattle, yak showed better growth performance. The serum biochemical results showed that the albumin/globulin ratio in yak serum was significantly higher than that in cattle. Aspartate aminotransferase, indirect bilirubin, creatine kinase, lactate dehydrogenase, and total cholesterol were significantly lower in yaks than in cattle. The rumen pH, acetate to propionate ratio, and acetate were lower in yaks than in cattle, whereas the lactate in yaks was higher than in cattle. There were significant differences in the structure of ruminal as well as fecal bacteria between cattle and yaks. The prediction of rumen bacterial function showed that there was a metabolic difference between cattle and yaks. In general, the metabolic pathway of cattle was mainly riched in a de novo synthesis of nucleotides, whereas that of yaks was mainly riched in the metabolic utilization of nutrients. This study provides a basis for understanding a rumen ecology under the condition of a high concentrate diet. Full article

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N¹⁰-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5′-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function. Full article

Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all. Full article

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2′-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2. Full article