Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Background/Aims: Recent studies revealed that patients with persistent aminotransferase elevations after antiviral treatment had higher risk of hepatic events; yet its underlying causes remain unclear. Our study aimed to investigate the etiologies of persistent aminotransferase elevations in patients treated with nucleos(t)ide analogs (NAs). Materials and Methods: A retrospective study was conducted on chronic hepatitis B (CHB) patients who had been receiving NA treatment for over a year and had an aminotransferase level greater than 40 IU/mL (more than twice, with a 3-month interval) and subsequently underwent a liver biopsy. Results: The study group included 46 patients (34 males) with a mean age of 44.8 ± 20.3 years (range: 24-71 years).The average dura- tion of NA therapy was 3.7 years (1.1-10.6 years). The etiologies of persistant transaminase elevation were categorized into 4 groups: patients with low hepatitis B virus (HBV) viral load (LVL, n = 11); concurrent non-alcoholic fatty liver disease (NAFLD, n = 12); concurrent other liver diseases (OLD, n = 12); and unknown liver dysfunction (ULD, n = 11). The proportion of G ≥ 2 inflammation was significantly higher in the LVL group (90.9%) compared to NAFLD (33.3%), OLD (50%), and ULD (27.2%) groups (P = .012). The hepatitis B e-antigen (HBeAg)-positive group exhibited a younger age (34.5 ± 10.2 vs. 48.1 ± 9.4 years, P < .001), a lower proportion of fibrosis F ≥ 2 (36.3% vs. 77.1%, P = .012), and a higher prevalence of detectable HBV DNA (54.5% vs.14.2%, P = .00632) compared to the HBeAg-negative group. Conclusion: The etiology of persistent aminotransferase elevations in CHB patients undergoing NAs treatment warrants investigation. Besides the commonly observed NAFLD and low HBV viral load, concurrent presence of other liver diseases requires elucidation.The proportion of G≥2 inflammation was higher in the LVL group.

Natural cyclic dinucleotide (CDN) is the secondary messenger involved in bacterial hemostasis, human innate immunity, and bacterial antiphage immunity. Synthetic CDN and its analogues are key molecular probes and potential immunotherapeutic agents. Several CDN analogues are under clinical research for antitumor immunotherapy. A myriad of synthetic methods have been developed and reported for the preparation of CDN and its analogues. However, most of the protocols require multiple steps, and only one CDN or its analogue is prepared at a time. In this study, a strategy based on a macrocyclic ribose phosphate skeleton containing a 1\'-alkynyl group was designed and developed to prepare CDN analogues containing triazolyl C-nucleosides by click chemistry. Combinatorial application of click chemistry and the sulfenylation cascade to the macrocyclic skeleton expanded the diversity of the CDN analogues. This macrocyclic skeleton strategy rapidly and efficiently provides CDN analogues to facilitate research on microbiology, immunology, and immunotherapy.

Herein, we report the synthesis of a new hybrid compound based on a 2\'-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2\'-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of \"non-typical\" minor products of the reaction. In addition to \"typical\" N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2\'-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2\'-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this \"upside down\" arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products.

1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/RACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(β-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-β-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.

Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogues used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of co-expressed ENT1. We created a cell model where ENT1 was removed from HEK293 cells using CRISPR/cas9 (ENT1KO cells); this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by qPCR. Wildtype HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 {plus minus} 0.06 and 1.50 {plus minus} 0.22 pmol/µl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 µM), while hypoxanthine was the only nucleobase with a sub-millimolar affinity (320 µM). A range of nucleoside/nucleobase analogues were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 µM for ticagrelor to 2,300 µM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. Significance Statement We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Global Natural Products Social (GNPS) molecular networking platform was applied to discovery the undescribed compounds from the common marine fungi Aspergillus versicolor CGF9-1-2, ultimately resulting in isolation of four new polyketides, decumbenone E (1), decumbenone F (2), 2\'-epi-8-O-methylnidurufin (6), (-)-phomoindene A (7), one new nucleoside, 3-methyl-9-(2-methylbutene)-xanthine (8), and five known analogues. Their structures were elucidated based on 1D/2D NMR spectroscopic and HRESIMS data analyses, meanwhile, the absolute configurations of new compounds were established based on the X-ray crystallographic experiments, as well as the electronic circular dichroism (ECD) analysis. All compounds were predicted pharmaceutical chemistry with ten commonly disease-related proteins by molecular docking. In addition, all compounds against TDP1 were performed in vitro, which was consistent with the docking result, and compound 6 shown a weak inhibitory activity.

Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today\'s nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5\'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the \"water problem\" that prohibits the synthesis of nucleotides in water.

Nucleosidic diarylethenes (DAEs) have evolved from an emerging class of photochromes into a well-established option for integrating photochromic functionalities into biological systems. However, a comprehensive understanding of how chemical structure influences their photochromic properties remains essential. While structural features, such as an inverse connection between the aryl residues and the ethene bridge, are well-documented for classical DAEs, their application to nucleosidic DAEs has been underexplored. In this study, we address this gap by developing three distinct types of inverse nucleosidic DAEs - semi-inverse thiophenes, semi-inverse uridines and inverse uridines. We successfully synthesized these compounds and conducted comprehensive analyses of their photostationary states, thermal stability, reversibility, and reaction quantum yields. Additionally, we conducted an in-depth comparison of their photochromic properties with those of their normal-type counterparts. Among the synthesized compounds, seven semi-inverse thiophenes exhibited the most promising characteristics. Notably, these compounds demonstrated excellent fatigue resistance, with up to 96% retention of photochromic activity over 40 switching cycles, surpassing the performance of all comparable nucleosidic DAEs reported to date. These findings hold significant promise for future applications in various fields.