Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8⁺ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8⁺ T cell responses and increasing the number of CD8⁺ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. Graphical Abstract

Transfer RNA (tRNA) modifications have emerged as critical post-transcriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications—the tRNA epitranscriptome—very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here, we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue would facilitate biomarker and drug discovery and development.

Purpose ¹⁸F-Alfatide II has been translated into clinical use and been proven to have good performance in identifying breast cancer. In this study, we investigated ¹⁸F-Alfatide II for evaluation of axillary lymph nodes (ALN) in breast cancer patients and compared the performance with ¹⁸F-FDG. Methods A total of 44 female patients with clinically suspected breast cancer were enrolled and underwent ¹⁸F-Alfatide II and ¹⁸F-FDG PET/CT within a week. Tracer uptakes in ALN were evaluated by visual analysis, semi-quantitative analysis with maximum standardized uptake value (SUV_max), mean standardized uptake value (SUV_mean), and SUV_max ratio of target/non-target (T/NT). Results Among 44 patients, 37 patients were pathologically diagnosed with breast cancer with metastatic (17 cases) or non-metastatic (20 cases) ALN. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of visual analysis were 70.6%, 90%, 81.1%, 85.7%, and 78.3% for ¹⁸F-Alfatide II, 64.7%, 90%, 78.4%, 84.6%, and 75% for ¹⁸F-FDG, respectively. By combining ¹⁸F-Alfatide II and ¹⁸F-FDG, the sensitivity significantly increased to 82.4%, the specificity was 85%, the accuracy increased to 83.8%, the PPV was 82.4%, and the NPV significantly increased to 85.0%. Three cases of luminal B subtype were false negative for both ¹⁸F-Alfatide II and ¹⁸F-FDG. The other 2 false negative cases of ¹⁸F-Alfatide II were triple-negative subtype and 3 false negative cases of ¹⁸F-FDG were luminal B subtype too. The AUCs of three semi-quantitative parameters (SUV_max, SUV_mean, T/NT) for ¹⁸F-Alfatide II were between 0.8 and 0.9, whereas those for ¹⁸F-FDG were more than 0.9. ¹⁸F-Alfatide II T/NT had the highest Youden index (76.5%), specificity (100%), accuracy (89.2%), and PPV (100%) among these semi-quantitative parameters. ¹⁸F-Alfatide II uptake as well as ¹⁸F-FDG uptake in metastatic axillary lymph nodes (MALN) was significantly higher than that in benign axillary lymph nodes (BALN). Both ¹⁸F-Alfatide II and ¹⁸F-FDG did not show difference in primary tumor uptake irrespective of ALN status. Conclusion ¹⁸F-Alfatide II can be used in breast cancer patients to detect metastatic ALN, however, like ¹⁸F-FDG, with high specificity but relatively low sensitivity. The combination of ¹⁸F-Alfatide II and ¹⁸F-FDG can significantly improve sensitivity and NPV. ¹⁸F-Alfatide II T/NT may serve as the most important semi-quantitative parameter to evaluate ALN.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA ^{Glu, Gln, Asp} contains a variety of xnm ⁵ s ² U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm ⁵ s ² U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm ⁵ s ² U to mnm ⁵ s ² U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm ⁵ s ² U in both Bacillus subtilis and Streptococcus mutans . This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm ⁵ s ² U into mnm ⁵ s ² U in B. subtilis . Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature. IMPORTANCE: The xnm ⁵ s ² U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm ⁵ s ² U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Queuosine (Q) is a naturally occurring modified nucleoside that occurs in the first position of transfer RNA anticodons such as Asp, Asn, His, and Tyr. As eukaryotes lack pathways to synthesize queuine, the Q nucleobase, they must obtain it from their diet or gut microbiota. Previously, we described the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica and characterized the enzyme EhTGT responsible for queuine incorporation into tRNA. At present, it is unknown how E. histolytica salvages queuine from gut bacteria. We used liquid chromatography–mass spectrometry (LC–MS) and N-acryloyl-3-aminophenylboronic acid (APB) PAGE analysis to demonstrate that E. histolytica trophozoites can salvage queuine from Q or E. coli K12 but not from the modified E. coli QueC strain, which cannot produce queuine. We then examined the role of EhDUF2419, a protein with homology to DNA glycosylase, as a queuine salvage enzyme in E. histolytica. We found that glutathione S-transferase (GST)-EhDUF2419 catalyzed the conversion of Q into queuine. Trophozoites silenced for EhDUF2419 expression are impaired in their ability to form Q-tRNA from Q or from E. coli. We also observed that Q or E. coli K12 partially protects control trophozoites from oxidative stress (OS), but not siEhDUF2419 trophozoites. Overall, our data reveal that EhDUF2419 is central for the direct salvaging of queuine from bacteria and for the resistance of the parasite to OS.

A three-color fluorescent molecular beacon assay for rapid profiling of substrate specificity of hOGG1 variants, and for engineering proteins to map genomic modifications.