A multi-omics investigation of the lung injury induced by PM2.5 at environmental levels via the lung-gut axis.

Long-term exposure to fine particulate matter (PM2.5) posed injury for gastrointestinal and respiratory systems, ascribing with the lung-gut axis. However, the cross-talk mechanisms remain unclear. Here, we attempted to establish the response networks of lung-gut axis in mice exposed to PM2.5 at environmental levels. Male Balb/c mice were exposed to PM2.5 (dose of 0.1, 0.5, and 1.0 mg/kg) collected from Chengdu, China for 10 weeks, through intratracheally instillation, and examined the effect of PM2.5 on lung functions of mice. The changes of lung and gut microbiota and metabolic profiles of mice in different groups were determined. Furthermore, the results of multi-omics were conjointly analyzed to elucidate the primary microbes and the associated metabolites in lung and gut responsible for PM2.5 exposure. Accordingly, the cross-talk network and key pathways between lung-gut axis were established. The results indicated that exposed to PM2.5 0.1 mg/kg induced obvious inflammations in mice lung, while emphysema was observed at 1.0 mg/kg. The levels of metabolites guanosine, hypoxanthine, and hepoxilin B3 increased in the lung might contribute to lung inflammations in exposure groups. For microbiotas in lung, PM2.5 exposure significantly declined the proportions of Halomonas and Lactobacillus. Meanwhile, the metabolites in gut including L-tryptophan, serotonin, and spermidine were up-regulated in exposure groups, which were linked to the decreasing of Oscillospira and Helicobacter in gut. Via lung-gut axis, the activations of pathways including Tryptophan metabolism, ABC transporters, Serotonergic synapse, and Linoleic acid metabolism contributed to the cross-talk between lung and gut tissues of mice mediated by PM2.5. In summary, the microbes including Lactobacillus, Oscillospira, and Parabacteroides, and metabolites including hepoxilin B3, guanosine, hypoxanthine, L-tryptophan, and spermidine were the main drivers. In this lung-gut axis study, we elucidated some pro- and pre-biotics in lung and gut microenvironments contributed to the adverse effects on lung functions induced by PM2.5 exposure.

Multi-omics reveals the testosterone promotion effect mechanism of Cordyceps Sobolifera on Leydig cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sobolifera (CS) has been traditionally utilized as an ethnic remedy for various health conditions, including chronic kidney diseases, anti-fatigue interventions, and management of chronic inflammation. Notably, CS is recognized for its substantial content of bioactive compounds, among which nucleosides prominently feature as constituents with diverse therapeutic advantages. AIM OF THE STUDY: This study aims to investigate the effects of CS on testosterone secretion in Leydig cells and explore the underlying mechanism. MATERIALS AND METHODS: Leydig cells were isolated from rat testes to establish a primary rat Leydig cells model. Cell proliferation and testosterone secretion were assessed via the methyl-piperidino-pyrazole (MTT) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Samples earmarked for RNA sequencing (RNA-Seq) analysis facilitated the identification of significantly differentially expressed genes (DEGs), and we conducted Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analyses. The veracity of our findings was validated through quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. RESULTS: The results showed that CS and guanosine could promote Leydig cell proliferation and bolster testosterone secretion. Our integrative analysis of metabolomics and transcriptomics has unveiled the potential mechanisms governing testosterone synthesis. Specifically, metabolomics has illuminated striking correlations within cholesterol metabolism, and bile secretion. Concurrently, transcriptomics has underscored the pivotal roles played by the cyclic adenosine monophosphate (cAMP) signaling pathway and steroid hormone biosynthesis. Furthermore, our investigation has demonstrated CS's aptitude in elevating the expression of proteins and genes. Notably, our findings have elucidated that these effects can be mitigated by protein kinase A (PKA) and adenylate cyclase (AC) specific inhibitors. CONCLUSION: This study delineates the cAMP-PKA pathways as plausible mechanisms underpinning the testosterone-enhancing properties of CS, with guanosine emerging as a fundamental bioactive constituent.

Tubular dysfunction impairs renal excretion of pseudouridine in diabetic kidney disease.

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.

Recent Advances in Adenosine-to-Inosine RNA Editing in Cancer.

RNA epigenetics, or epitranscriptome, is a growing group of RNA modifications historically classified into two categories: RNA editing and RNA modification. RNA editing is usually understood as post-transcriptional RNA processing (except capping, splicing and polyadenylation) that changes the RNA nucleotide sequence encoded by the genome. This processing can be achieved through the insertion or deletion of nucleotides or deamination of nucleobases, generating either standard nucleotides such as uridine (U) or the rare nucleotide inosine (I). Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent type of RNA modification in mammals and is catalyzed by adenosine deaminase acting on the RNA (ADAR) family of enzymes that recognize double-stranded RNAs (dsRNAs). Inosine mimics guanosine (G) in base pairing with cytidine (C), thereby A-to-I RNA editing alters dsRNA secondary structure. Inosine is also recognized as guanosine by the splicing and translation machineries, resulting in mRNA alternative splicing and protein recoding. Therefore, A-to-I RNA editing is an important mechanism that causes and regulates "RNA mutations" in both normal physiology and diseases including cancer. In this chapter, we reviewed current paradigms and developments in the field of A-to-I RNA editing in the context of cancer.

Identification of Bona Fide RNA Editing Sites: History, Challenges, and Opportunities.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of enzymes of which there are three members (ADAR1, ADAR2, and ADAR3), is a major gene regulatory mechanism that diversifies the transcriptome. It is widespread in many metazoans, including humans. As inosine is interpreted by cellular machineries mainly as guanosine, A-to-I editing effectively gives A-to-G nucleotide changes. Depending on its location, an editing event can generate new protein isoforms or influence other RNA processing pathways. Researchers have found that ADAR-mediated editing performs diverse functions. For example, it enables living organisms such as cephalopods to adapt rapidly to fluctuating environmental conditions such as water temperature. In development, the loss of ADAR1 is embryonically lethal partly because endogenous double-stranded RNAs (dsRNAs) are no longer marked by inosines, which signal "self", and thus cause the melanoma differentiation-associated protein 5 (MDA5) sensor to trigger a deleterious interferon response. Hence, ADAR1 plays a key role in preventing aberrant activation of the innate immune system. Furthermore, ADAR enzymes have been implicated in myriad human diseases. Intriguingly, some cancer cells are known to exploit ADAR1 activity to dodge immune responses. However, the exact identities of immunogenic RNAs in different biological contexts have remained elusive. Consequently, there is tremendous interest in identifying inosine-containing RNAs in the cell.The identification of A-to-I RNA editing sites is dependent on the sequencing of nucleic acids. Technological and algorithmic advancements over the past decades have revolutionized the way editing events are detected. At the beginning, the discovery of editing sites relies on Sanger sequencing, a first-generation technology. Both RNA, which is reverse transcribed into complementary DNA (cDNA), and genomic DNA (gDNA) from the same source are analyzed. After sequence alignment, one would require an adenosine to be present in the genome but a guanosine to be detected in the RNA sample for a position to be declared as an editing site. However, an issue with Sanger sequencing is its low throughput. Subsequently, Illumina sequencing, a second-generation technology, was invented. By permitting the simultaneous interrogation of millions of molecules, it enables many editing sites to be identified rapidly. However, a key challenge is that the Illumina platform produces short sequencing reads that can be difficult to map accurately. To tackle the challenge, we and others developed computational workflows with a series of filters to discard sites that are likely to be false positives. When Illumina sequencing data sets are properly analyzed, A-to-G variants should emerge as the most dominant mismatch type. Moreover, the quantitative nature of the data allows us to build a comprehensive atlas of editing-level measurements across different biological contexts, providing deep insights into the spatiotemporal dynamics of RNA editing. However, difficulties remain in identifying true A-to-I editing sites in short protein-coding exons or in organisms and diseases where DNA mutations and genomic polymorphisms are prevalent and mostly unknown. Nanopore sequencing, a third-generation technology, promises to address the difficulties, as it allows native RNAs to be sequenced without conversion to cDNA, preserving base modifications that can be directly detected through machine learning. We recently demonstrated that nanopore sequencing could be used to identify A-to-I editing sites in native RNA directly. Although further work is needed to enhance the detection accuracy in single molecules from fewer cells, the nanopore technology holds the potential to revolutionize epitranscriptomic studies.

Biodegradation of Inosine and Guanosine by Bacillus paranthracis YD01.

Both inosine and guanosine are precursors of uric acid that may cause the diseases of hyperuricemia and gout in humans. Here, a promising bacterial strain for efficiently biodegrading both inosine and guanosine was successfully isolated from a healthy human intestine and identified as Bacillus paranthracis YD01 with 16S rRNA analysis. An initial amount of 49.6 mg·L-1 of inosine or 49.9 mg·L-1 of guanosine was completely removed by YD01 within 12 h, which showed that YD01 had a strong ability to biodegrade inosine and guanosine. Furthermore, the initial amount of 49.2 mg·L-1 of inosine or 49.5 mg·L-1 of guanosine was totally catalyzed by the intracellular crude enzymes of YD01 within 6 h, and the initial inosine amount of 49.6 mg·L-1 or guanosine of 49.7 mg·L-1 was biodegraded by the extracellular crude enzymes of YD01 within 9 h. Illumina Hiseq sequencing and database gene annotation were used to elucidate the genomic characteristics of B. paranthracis YD01. Purine nucleoside phosphorylase, encoded by gene 1785, gene 3933, and gene 4403, was found in the KEEG database, which played a crucial role in the biodegradation of inosine and guanosine. The results of this study provide valuable insights into the mechanisms for biodegrading inosine and guanosine using B. paranthracis YD01.

Compatibility of Nucleobases Containing Pt(II) Complexes with Red Blood Cells for Possible Drug Delivery Applications.

The therapeutic advantages of some platinum complexes as major anticancer chemotherapeutic agents and of nucleoside analogue-based compounds as essential antiviral/antitumor drugs are widely recognized. Red blood cells (RBCs) offer a potential new strategy for the targeted release of therapeutic agents due to their biocompatibility, which can protect loaded drugs from inactivation in the blood, thus improving biodistribution. In this study, we evaluated the feasibility of loading model nucleobase-containing Pt(II) complexes into human RBCs that were highly stabilized by four N-donors and susceptible to further modification for possible antitumor/antiviral applications. Specifically, platinum-based nucleoside derivatives [PtII(dien)(N7-Guo)]2+, [PtII(dien)(N7-dGuo)]2+, and [PtII(dien)(N7-dGTP)] (dien = diethylenetriamine; Guo = guanosine; dGuo = 2'-deoxy-guanosine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate) were investigated. These Pt(II) complexes were demonstrated to be stable species suitable for incorporation into RBCs. This result opens avenues for the possible incorporation of other metalated nucleobases analogues, with potential antitumor and/or antiviral activity, into RBCs.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Screening and application of purine degrading Limosilactobacillus fermentum LF-1 from Huangjiu fermentation broth.

BACKGROUND: As the important building blocks of nucleic acids, purines are alkaloids and responsible for hyperuricemia and gout. The purine content in Huangjiu is higher, and mainly exists in the form of free bases, which is easier to be absorbed by human body. However, the currently available reports on purine in Huangjiu mainly focus on detection methods and content survey. No studies on the regulation of the purine content in Huangjiu have been reported. RESULTS: Eighty-four strains, with the degradation capacity of purine, were screened from the fermentation broth of Huangjiu. In detail, the isolated lactic acid bacteria (LAB) strain 75 # showed the strongest degradation ability of guanosine, inosine and four purines, which reduce their levels by 83.4% (guanosine), 97.4% (inosine), 95.1% (adenine), 95.0% (guanine), 94.9% (hypoxanthine) and 65.9% (xanthine), respectively. Subsequently, the LAB strain 75# was identified to be Limosilactobacillus fermentum by 16S rRNA gene sequencing, which was named as Limosilactobacillus fermentum LF-1 and applied to the fermentation of Huangjiu in the laboratory. Compared with the fermentation broth of Huangjiu without adding L. fermentum LF-1, the content of purine compounds in the fermentation broth inoculated with L. fermentum LF-1 was reduced by 64.7%. In addition, the fermented Huangjiu had richer flavor compounds, and the physicochemical indices were in accordance with the national standard of Chinese Huangjiu. CONCLUSION: The screened strain L. fermentum LF-1 may be a promising probiotic for the development of a novel that can efficiently degrade purine in Huangjiu. © 2023 Society of Chemical Industry.

Blocking tri-methylguanosine synthase 1 (TGS1) stops anchorage-independent growth of canine sarcomas.

Tri methylguanosine synthase 1 (TGS1) is the enzyme that hyper methylates the hallmark 7-methyl-guanosine cap (m7G-cap) appended to the transcription start site of RNAs. The m7G-cap and the eIF4E-cap binding protein guide canonical cap-dependent translation of mRNAs, whereas hyper methylated cap, m2,2,7G-cap (TMG) lacks adequate eIF4E affinity and licenses entry into a different translation initiation pathway. The potential role for TGS1 and TMG-capped mRNA in neoplastic growth is unknown. Canine sarcoma has high translational value to the human disease. Cumulative downregulation of protein synthesis in osteosarcoma OSCA-40 was achieved cooperatively by siTGS1 and Torin-1. Torin-1 inhibited the proliferation of three canine sarcoma explants in a reversible manner that was eliminated by siRNA-downregulation of TGS1. TGS1 failure prevented the anchorage-independent growth of osteo- and hemangio-sarcomas and curtailed sarcoma recovery from mTOR inhibition. RNA immunoprecipitation studies identified TMG-capped mRNAs encoding TGS1, DHX9 and JUND. TMG-tgs1 transcripts were downregulated by leptomycin B and TGS1 failure was compensated by eIF4E mRNP-dependent tgs1 mRNA translation affected by mTOR. The evidence documents TMG-capped mRNAs are hallmarks of the investigated neoplasms and synergy between TGS1 specialized translation and canonical translation is involved in sarcoma recovery from mTOR inhibition. Therapeutic targeting of TGS1 activity in cancer is ripe for future exploration.

Sports Participation Promotes Beneficial Adaptations in the Erythrocyte Guanylate Nucleotide Pool in Male Athletes Aged 20-90 Years.

Introduction: The guanine nucleotide pool (GTP, guanosine-5'-triphosphate; GDP, guanosine-5'-diphosphate, and GMP, guanosine-5'-monophosphate) is an essential energy donor in various biological processes (eg protein synthesis and gluconeogenesis) and secures several vital regulatory functions in the human body. The study aimed to predict the trends of age-related changes in erythrocyte guanine nucleotides and examine whether competitive sport and related physical training promote beneficial adaptations in erythrocyte guanylate concentrations. Methods: The study included 86 elite endurance runners (EN) aged 20-81 years, 58 sprint-trained athletes (SP) aged 21-90 years, and 62 untrained individuals (CO) aged 20-68 years. Results: The concentration of erythrocyte GTP and total guanine nucleotides (TGN) were highest in the SP group, lower in the EN group, and lowest in the CO group. Both athletic groups had higher guanylate energy charge (GEC) values than the CO group (p = 0.012). Concentrations of GTP, TGN, and GEC value significantly decreased, while GDP and GMP concentrations progressively increased with age. Conclusion: Such a profile of change suggests a deterioration of the GTP-related regulatory function in older individuals. Our study explicitly shows that lifelong sports participation, especially of sprint-oriented nature, allows for maintaining a higher erythrocyte guanylate pool concentration, supporting cells' energy metabolism, regulatory and transcription properties, and thus more efficient overall body functioning.

Metabolic effects of radiation on red blood cells from cold stored whole blood.

BACKGROUND: The risk of military and civilian radiation exposure is increasing, and determining the effects of exposure is a high priority. Irradiation of the nearby blood supply after a nuclear event may impede mobilization of blood products for resuscitation at a time of great need. RBCs are administered to patients with trauma and hemorrhage to transport and deliver oxygen and avoid tissue hypoxia. Here we determine the effects of ionizing radiation on the energy metabolome of RBCs isolated from cold stored whole blood to determine if their stability is compromised by radiation exposure. STUDY DESIGN AND METHODS: Whole blood from healthy volunteers was subjected to 0, 25, or 75 Gy of X-irradiation, and stored at 4°C. RBCs were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. The levels of extracted Krebs cycle intermediates, nicotinamide adenine dinucleotides, and phosphorylated derivatives of adenosine and guanosine were determined by tandem mass spectroscopy. RESULTS: Irradiation at either 25Gy or 75Gy had no significant effect on any parameter measured compared to control (0Gy). However, there was a significant change over time in storage for ATP, GDP, and guanosine. DISCUSSION: Irradiation at doses up to 75Gy had no effect on the energy metabolome of RBCs prepared from blood stored at 4°C for up to 21 days, suggesting that the RBC energy metabolome is not affected by radiation exposure and the blood can still be used for resuscitation in trauma patients.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Direct Measurement of Nucleoside Ribohydrolase Enzyme Activities in Trichomonas vaginalis Cells Using 19F and 13C-Edited 1H NMR Spectroscopy.

Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis. Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. Since T. vaginalis is an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use in T. vaginalis cells. The 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13C-editing to resolve the substrate 1H signals from cell and growth media background signals. The in-cell NMR assays are robust and have been demonstrated to provide inhibition data on test compounds. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.

NMDA receptor-mediated modulation on glutamine synthetase and glial glutamate transporter GLT-1 is involved in the antidepressant-like and neuroprotective effects of guanosine.

Guanosine has been reported to elicit antidepressant-like responses in rodents, but if these actions are associated with its ability to afford neuroprotection against glutamate-induced toxicity still needs to be fully understood. Therefore, this study investigated the antidepressant-like and neuroprotective effects elicited by guanosine in mice and evaluated the possible involvement of NMDA receptors, glutamine synthetase, and GLT-1 in these responses. We found that guanosine (0.05 mg/kg, but not 0.01 mg/kg, p. o.) was effective in producing an antidepressant-like effect and protecting hippocampal and prefrontocortical slices against glutamate-induced damage. Our results also unveiled that ketamine (1 mg/kg, but not 0.1 mg/kg, i. p, an NMDA receptor antagonist) effectively elicited antidepressant-like actions and protected hippocampal and prefrontocortical slices against glutamatergic toxicity. Furthermore, the combined administration of sub-effective doses of guanosine (0.01 mg/kg, p. o.) with ketamine (0.1 mg/kg, i. p.) promoted an antidepressant-like effect and augmented glutamine synthetase activity and GLT-1 immunocontent in the hippocampus, but not in the prefrontal cortex. Our results also showed that the combination of sub-effective doses of ketamine and guanosine, at the same protocol schedule that exhibited an antidepressant-like effect, effectively abolished glutamate-induced damage in hippocampal and prefrontocortical slices. Our in vitro results reinforce that guanosine, ketamine, or sub-effective concentrations of guanosine plus ketamine protect against glutamate exposure by modulating glutamine synthetase activity and GLT-1 levels. Finally, molecular docking analysis suggests that guanosine might interact with NMDA receptors at the ketamine or glycine/d-serine co-agonist binding sites. These findings provide support for the premise that guanosine has antidepressant-like effects and should be further investigated for depression management.

Arabidopsis DXO1 activates RNMT1 to methylate the mRNA guanosine cap.

Eukaryotic messenger RNA (mRNA) typically contains a methylated guanosine (m7G) cap, which mediates major steps of mRNA metabolism. Recently, some RNAs in both prokaryotic and eukaryotic organisms have been found to carry a non-canonical cap such as the NAD cap. Here we report that Arabidopsis DXO family protein AtDXO1, which was previously known to be a decapping enzyme for NAD-capped RNAs (NAD-RNA), is an essential component for m7G capping. AtDXO1 associates with and activates RNA guanosine-7 methyltransferase (AtRNMT1) to catalyze conversion of the guanosine cap to the m7G cap. AtRNMT1 is an essential gene. Partial loss-of-function mutations of AtRNMT1 and knockout mutation of AtDXO1 reduce m7G-capped mRNA but increase G-capped mRNAs, leading to similar pleiotropic phenotypes, whereas overexpression of AtRNMT1 partially restores the atdxo1 phenotypes. This work reveals an important mechanism in m7G capping in plants by which the NAD-RNA decapping enzyme AtDXO1 is required for efficient guanosine cap methylation.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

A non-carboxylating pentose bisphosphate pathway in halophilic archaea.

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1,5-bisphosphate (R15P) isomerase and ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco. In this study, we identify a previously unrecognized nucleoside degradation pathway in halophilic archaea, composed of guanosine phosphorylase, ATP-dependent ribose-1-phosphate kinase, R15P isomerase, RuBP phosphatase, ribulose-1-phosphate aldolase, and glycolaldehyde reductase. The pathway converts the ribose moiety of guanosine to dihydroxyacetone phosphate and ethylene glycol. Although the metabolic route from guanosine to RuBP via R15P is similar to that of the pentose bisphosphate pathway in Thermococcales, the downstream route does not utilize Rubisco and is unique to halophilic archaea.

The Importance of Toll-like Receptor 9 Expression on Monocytes and Dendritic Cells in the Context of Epstein-Barr Virus Infection in the Immunopathogenesis of Primary Glomerulonephritis.

Toll-like receptor 9 (TLR9) is activated by unmethylated cytosine-phosphate-guanosine (CpG) dinucleotides found in the genomes of pathogens such as Epstein-Barr virus (EBV). The aim of this study was to determine the role of TLR9 in the immunopathogenesis of IgA nephropathy (IgAN) and membranoproliferative glomerulonephritis (MPGN) in the context of Epstein-Barr virus (EBV) infection. For this purpose, the frequency of TLR9-positive monocytes and dendritic cells (DCs, i.e., BDCA-1; myeloid dendritic cells, and BDCA-2; plasmocytoid dendritic cells) was studied, and a quantitative analysis of the concentration of TLR9 in the serum of patients diagnosed with IgAN and MPGN was undertaken. Higher frequencies of TLR9-positive DCs and monocytes in IgAN and MPGN patients were observed as compared with the control group. Patients diagnosed with GN exhibited a higher percentage of BDCA-1+CD19- and BDCA-2+CD123+ DCs than patients in the control group. Moreover, serum TLR9 concentration was shown to be significantly correlated with EBV DNA copy number/µg DNA, IgG, IgM, serum albumin, total protein in 24-h urine collection test and the frequency of BDCA-2+CD123+ DCs in peripheral blood. Our findings confirm that TLR9 may be involved in the development of IgAN and MPGN.