Anthocyanins are a subgroup of flavonoid polyphenols previously investigated for improving cardiovascular health and preventing the development of endothelial dysfunction. However, their poor bioavailability raises the question of whether the observed biological activity is due to their metabolites. Phenolic metabolites can reach higher plasma concentrations and can persist in the circulation for periods much longer than their original anthocyanin form; therefore, the biological activity and health promoting effects of anthocyanins may differ from their metabolites. To address this, recent studies have facilitated different cell models, in vivo studies and explored physiologically relevant concentrations to better understand their mechanisms of action. The criteria were chosen based on previous reports demonstrating that anthocyanins can improve endothelial function via modulation of the Akt-endothelial nitric oxide synthase pathway and transcription factors Nrf2 and NF-κB, which made it critical to assess the phenolic metabolites’ modes of action via these pathways. This review demonstrates how phenolic metabolites differ in bioactivity from their precursor anthocyanin, demonstrating improved endothelial function in response to inflammatory mediators at concentrations that are tolerated in vivo. The review highlights the crucial need for further studies to focus on improving the bioavailability of metabolites in isolation and explore the effect of metabolites in mixtures.

Background/Objectives: Reduced nitric oxide (NO) bioavailability secondary to excess-superoxide-driven oxidative stress is central to endothelial dysfunction. Previous studies suggest that phenolic metabolites may improve NO bioavailability, yet limited research is available in response to an inflammatory mediator. Therefore, we assessed the effects of cyanidin-3-glucoside (C3G) and its phenolic metabolites protocatechuic acid (PCA) and vanillic acid (VA) on NO bioavailability in a TNF-α induced inflammatory environment. Methods: Primary human umbilical vein endothelial cells (HUVECs) were supplemented with either C3G, PCA, or VA at 1 μM for 24 h before being stimulated with TNF-α 20 ng/mL for an additional 24 h. Measurements included cell viability, apoptosis, reactive oxygen species (ROS), nitrite concentrations, and endothelial nitric oxide synthase (eNOS) and Akt at the mRNA and protein level. Results: Phenolic metabolites did not increase the eNOS expression or nitrite levels in the unstimulated environment; rather, the metabolites mediated NO bioavailability in response to TNF-α induced oxidative stress, with increased viability, eNOS mRNA, phosphorylation, and nitrite levels. Conclusions: Phenolic metabolites, in the presence of TNF-α, can improve NO bioavailability at physiologically relevant concentrations via the Akt-eNOS pathway. This demonstrates that the induction of inflammation is a prerequisite for phenolic metabolites to promote protective properties in endothelial cells by activating the Akt-eNOS pathway.

Background/Objectives: The large size of commonly used regulatory elements such as the cytomegalovirus (CMV) immediate-early promoter imposes a significant burden on the already restricted payload capacity of first-generation adenoviral vectors, potentially hindering the development of multi-antigen vaccine candidates. To address this limitation, we have engineered a panel of novel, small, semi-synthetic promoters designed to leverage the changes in transcriptomic milieu following adenoviral vector entry. Methods: Eight synthetic enhancer modules (SE1–SE8) were designed in silico, each composed of transcription factor binding sites (TFBSs) previously found in host genes that are upregulated during early adenoviral infection. These synthetic enhancers were coupled with a minimal CMV core promoter to generate a panel of compact semi-synthetic promoters (cSE1–cSE8), and their activity was evaluated in the context of ChAdOx1 viral vectors expressing GFP or a modified Plasmodium falciparum circumsporozoite (CSN) antigen. Promoter performance was characterised in vitro via flow cytometry, RT-qPCR, and Western blotting, and in vivo by quantifying antigen-specific T-cell (IFN-γ ELISpot) and IgG antibody (ELISA) responses in BALB/c mice. Results: In vitro characterisation revealed a wide range of promoter activity across the panel, with cSE3 and cSE5 driving transgene expression levels comparable to the benchmark CMV promoters despite their markedly reduced genomic footprint. In vivo, ChAdOx1 vectors incorporating cSE3 and cSE5 elicited potent antigen-specific T-cell and IgG responses that were comparable to those induced by the larger CMV control promoters. Conclusions: We have successfully developed semi-synthetic promoters that match the potency of the much larger, frequently used CMV promoters whilst simultaneously reducing genomic footprint. These novel regulatory elements will facilitate the design of next-generation vaccines, particularly those requiring large antigens or multi-antigen cassettes.

Messenger RNA (mRNA) therapeutics formulated in lipid nanoparticles (LNPs) have transformed the field of nucleic acid medicine by enabling transient, programmable protein expression for vaccination, protein replacement, immunotherapy, and genome engineering. Extending their clinical success beyond vaccines requires a mechanistic understanding of where LNP components and mRNA travel, which cells they enter, where cargo is translated, and how each component is cleared. Such biodistribution outcomes are governed by a complex interplay between nanoparticle physicochemical properties, administration route, protein corona formation, host physiology, intracellular trafficking, and degradation. Importantly, the biodistribution of lipid components, intact mRNA, degraded RNA fragments, and translated proteins represents distinct yet interconnected biological processes that should not be interpreted interchangeably. Advances in molecular imaging, quantitative nucleic acid analysis, mass spectrometry, spatial and single‐cell technologies, and pharmacokinetic modeling are improving their resolution across tissues and time. In parallel, rational lipid engineering, selective organ targeting, biodegradable ionizable lipids, ligand‐directed delivery, and artificial intelligence‐assisted design are expanding opportunities for tissue‐specific delivery beyond the liver and lymphoid organs. However, interpretation remains constrained by assay heterogeneity, incomplete understanding of protein corona dynamics and endosomal escape, species‐dependent differences, and the absence of harmonized regulatory standards. This review examines the mechanisms governing mRNA‐LNP disposition, evaluates what current analytical methods detect and miss, synthesizes preclinical and clinical evidence, and considers the strategies needed to achieve predictable, tissue‐selective, and clinically translatable mRNA delivery.

Pre-erythrocytic malaria vaccines aim to prevent infection at the earliest stage of parasite entry by blocking Plasmodium falciparum sporozoites before hepatocyte invasion, thereby halting progression to symptomatic blood-stage disease. The circumsporozoite protein (CSP), the dominant surface antigen of sporozoites, remains the leading target for pre-erythrocytic vaccine development. Here, we investigated the use of an mRNA vaccine platform to express CSP and examined how antigen localisation influences immunogenicity and protective efficacy. We compared mRNA constructs encoding soluble CSP with versions engineered for membrane anchoring using either a mammalian glycosylphosphatidylinositol (GPI) sequence derived from placental alkaline phosphatase or a heterologous transmembrane (TM) domain from influenza H1N1 haemagglutinin. Immunisation with CSP mRNA-LNP formulations showed that membrane association altered the magnitude and profile of antibody responses, with distinct effects observed across the NANP repeat and C-terminal regions of CSP. Following intravenous challenge with transgenic Plasmodium berghei sporozoites expressing P. falciparum CSP, both soluble and membrane-associated vaccines delayed parasitaemia and conferred partial protection, but membrane targeting did not consistently improve protective efficacy. These findings show that antigen localisation can shape the immune response to mRNA-expressed CSP, while also highlighting that enhanced immunogenicity does not necessarily translate into greater protection.