Abstract mRNA–lipid nanoparticle (LNP) vaccines are detectable in human blood after vaccination, but platform-specific differences in systemic persistence and transcript integrity remain poorly defined. We analyzed serial blood samples from 73 participants receiving Moderna mRNA-1273 (three formulations), Pfizer/BioNTech BNT162b2, or an investigational receptor-binding domain (RBD) mRNA vaccine (three different doses). Using droplet digital polymerase chain reaction (ddPCR) assays, we quantified total and long-range linked (“intact”) vaccine mRNA, and we measured vaccine-specific ionizable lipids by liquid chromatography–mass spectrometry (LC–MS). Across platforms, mRNA decay was fastest for mRNA-1273, intermediate for BNT162b2, and slowest for the RBD vaccine, with ionizable lipid decay following the same rank order. Notably, intact spike mRNA declined two-fold faster after mRNA-1273 than BNT162b2 vaccination. Kinetics modelling revealed platform-dependent coupling of mRNA and lipid kinetics: intact mRNA tracked closely with SM-102 for mRNA-1273, whereas ALC-0315 persisted longer than intact mRNA for BNT162b2. A ten-fragment linkage ddPCR panel spanning the spike transcript showed lower linkage toward 3′-proximal regions that mirrored the administered mRNA-1273 formulation. Together, these data establish a quantitative framework for benchmarking mRNA–LNP platform kinetics and transcript integrity in humans.

Shellfish allergy is a major global health concern, with prawns representing the most common trigger. Black tiger prawn (Penaeus monodon, BTP) and white leg prawn (Litopenaeus vannamei, WLP) dominate global consumption. Here, label-free shotgun proteomics combined with in-silico allergenicity prediction was applied to identify and compare the allergen profiles of BTP and WLP. Raw prawn extracts were analyzed by LC-MS/MS, and allergens were identified and predicted using AllerCatPro. The relative abundance of allergens was assessed using iBAQ%, and the differential abundance was evaluated using LFQ intensity. Although allergens accounted for only ∼4% of identified proteins, they represented 34-38% of total protein abundance, highlighting their immunological relevance. Myosin light chain was the most abundant allergen, followed by arginine kinase, sarcoplasmic calcium-binding protein, and tropomyosin in both species. Four low-abundance proteins were identified as novel allergen candidates. Notable differences in allergen profiles between the two species were observed at the level of allergen orthologs, isoforms, and variants, with nine uniquely identified in BTP and seven in WLP. Together, these findings reveal species-specific variations with implications for improved diagnostic strategies, therapeutic approaches, and allergen detection methods for shellfish allergy.

Type 2 diabetes and obesity are commonly accompanied by metabolic dysfunction-associated steatotic liver disease (MASLD), increasing the risk of developing metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis. The early stages of MASLD are characterized by dysfunctional lipid metabolism, including remodelling of the hepatic lipidome. In this context, reductions in hepatic phosphatidylserine (PS) have been associated with increased hepatic steatosis, inflammation and fibrosis. In this study, we investigated the impact of dietary PS supplementation on liver function and systemic metabolic homeostasis in mice with hepatic steatosis and MASH. Taking advantage of the MUP-uPA mouse model, including wild-type (WT) mice with hepatic steatosis and MUP-uPA mice with MASH and fibrosis, we show that PS supplementation reduces hepatic triglyceride accumulation, inflammation and fibrosis in male MUP-uPA mice. Supporting these data, PS supplementation suppresses fibrogenic gene expression in LX-2 hepatic stellate cells. We further show that PS supplementation improves glycaemic control and insulin sensitivity in male and female mice, which is associated with enhanced insulin signalling in muscle and liver, despite a pronounced suppression of glycolysis, glucose oxidation and glycogen breakdown in liver, muscle and/or adipose tissue. Metabolic flux analysis suggests a shift in substrate utilization, favouring fatty acid metabolism, particularly in muscle, while further pointing to marked improvements in mitochondrial function and oxidative capacity. These findings indicate that PS exerts multifaceted benefits by improving both MASH and whole-body glucose homeostasis, independent of conventional oxidative glucose metabolism. Our results support further investigation into dietary PS as a potential complementary strategy for MASH and glycaemic control.

Allergic reactions to fish pose complex food safety challenges, driven by species diversity and underexplored intraspecies variability. This study comparatively examines allergen profiles in Malabar red snappers (Lutjanus malabaricus, n = 39) across body sizes, anatomical regions and production origins using SDS-PAGE, immunoblotting and quantitative mass spectrometry. Protein profiles varied greatly by fish size and muscle region, but not by origin. Smaller fish contained higher levels of major allergen parvalbumin and creatine kinase, while larger fish exhibited elevated levels of heat-labile allergens enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase. Parvalbumin levels were highest in head, followed by belly, dorsal, and tail. Greatest variation was observed for the three heat-stable allergens - parvalbumin, tropomyosin, and collagen. Minimal origin-dependent differences affected 2 of 11 registered fish allergens. We established an integrated proteomics workflow for systematic allergenicity assessments to uncover intraspecies variability and provide foundational knowledge for understanding intraspecies variability to improve food safety strategies.

The Gram-negative oral pathogen, Porphyromonas gingivalis, uses the Type IX Secretion System (T9SS) to secrete major virulence factors (cargo proteins) and anchor them to the cell surface via a novel linking sugar, 2-N-seryl, 3-N-acetylglucuronamide (SAGA), which is a component of a specific type of lipopolysaccharide, A-LPS. The reported structure of the polysaccharide component (A-PS) was a repeating phosphorylated mannan whereas the PS of conventional O-LPS (O-PS) is a repeating Gal-Glu-Rha-GalNAc unit. Here, we have performed extensive mass spectrometric analyses of cargo protein-linked LPS with and without proteinase K treatment to determine the structure of A-LPS. Limited acid hydrolysis of the PS backbone with trifluoromethanesulfonic acid enabled long PS fragments linked to cargo-derived peptides to be identified for the first time. Unexpectedly, rather than finding A-PS units, up to eleven O-PS repeating units were found linked to cargo via a novel pentasaccharide linker designated A-LS, composed of SAGA-Hex-dHex(C4H4O3)(Pent)-Hex. In addition, samples from a wzzP/porT double mutant that produced free truncated O-PS were specifically hydrolyzed to cleave lipid A prior to MS analysis. In these samples A-LS was found attached to a limited number of O-PS repeating units that in turn were associated with a putative core oligosaccharide that included the LPS-specific sugar, 3-deoxy-d-manno-octulosonic acid (Kdo). The proposed structure of A-LPS explains all 11 genes specific to A-LPS biosynthesis, and provides the first structural evidence that cargo proteins such as the gingipains are anchored to the cell surface via a complete LPS molecule.

Seafood allergy is complex due to extensive species diversity, posing major challenges in food safety assessments, clinical diagnosis and dietary management. However, the absence of established workflows to resolve allergenomes limits correlations between allergen abundance, clinical sensitisation, and consumer risk. Mass spectrometry (MS)-based proteomics overcomes limitations of conventional immunoassay allergen detection by enabling unbiased protein identification and quantification, including allergen isoforms and low-abundance proteins within complex matrices. An integrated workflow combining immunological analyses, liquid chromatography-MS/MS proteomics, and bioinformatics was developed to characterise allergenomes across eight commonly consumed Asia-Pacific fish species. Comprehensive allergen profiles were established using in silico allergenicity predictions with AllerCatPro, combined with immunological validations using allergen-specific antibody and pooled patient sera. Across eight species, 529-1012 protein groups were identified, including all 11 fish muscle allergens, with pronounced interspecies differences in allergen composition and isoform distribution. The major fish pan-allergen parvalbumin was the most abundant allergen of most species and varied in abundance by up to 7-fold. Mackerel displayed a distinct low-parvalbumin profile with enriched metabolic allergens. Tissue heating induced a consistent shift toward enrichment of heat-stable and tissue-retained allergens, particularly parvalbumin, tropomyosin, and collagen. In contrast, heating of raw extracts generated more variable and species-specific retention of selected proteins, including heat-labile metabolic enzymes. In silico analysis predicted 14 proteins with strong allergenicity evidence for further validation. Fish allergenomes are species-specific and processing-dependent, positioning quantitative proteomics as a powerful platform for improved molecular risk assessment, and the development of representative diagnostic and food safety reference materials.

Fucoidan and laminarin are well-recognized for their potent bioactivities. However, the influence of their structural characteristics on antioxidative activity and the mechanism underlying their antidiabetic effects remains underexplored. This study aims to investigate the relationship between their structural features and bioactive performance. Additionally, the study seeks to rationalize their alpha-glucosidase inhibitory mechanism through molecular docking analysis. As discovered, they differed primarily in monosaccharide composition, molecular weight (Mw), and sulfate content. Fucoidans exhibited stronger DPPH and hydroxyl radical scavenging activities, while laminarins showed greater alpha-glucosidase inhibitory effects. These differences may be attributed to the higher Mw and sulfation degree of fucoidans. Molecular docking analysis of representative polysaccharide fragments indicates that hydrogen bonding plays a key role in their strong binding affinity to alpha-glucosidase, suggesting a potential mechanism for their enzyme-inhibitory activity. This research offers insight into structural traits influencing their bioactivities, guiding future efforts to optimize the corresponding functional properties.

Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5'-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial, dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple aminoacyl-tRNA synthetases (aaRSs), including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.

Background and Methods: Using a nanoparticle-based enrichment (Proteonano) methodology on human plasma samples, we achieved a substantial increase in identified proteins from ~700 to >5000 proteins compared to neat plasma digest. In a small-scale pilot test, we applied this methodology to a small cohort of plasma samples from pancreatic cancer (PC) patients with different disease stages: (I) primary tumor and (II) metastases, and compared them with healthy controls. Most identified proteins are within the Human Plasma Proteome Project (HPPP) database, and more than 300 proteins are on the list of FDA-approved drug targets. Results: We observed a large and significant increase in ribosomal proteins in the plasma of patients with metastatic PC. ADH1C and ADH1B, both members of the alcohol dehydrogenase family, were particularly upregulated in patients with liver metastasis. Fifteen other predicted secreted and/or cell surface–associated proteins with known cancer associations are also significantly altered and would otherwise go undetected in neat, digested plasma. Conclusions: The significant increase in proteome depth allows a strong foundation for future large-scale experimental and comparative analysis. Lastly, similar conclusions could be reached from comparing different mass spectrometers (Orbitrap Astral and Orbitrap Ascend) and columns (depth and throughput) setups on the same dataset, although the depth approach on the newer Orbitrap Astral instrumentations can reveal additional insights in the plasma proteome.

The effects of varying intensities of Australian Synchrotron source terahertz (THz) radiation on pheochromocytoma (PC 12) neuronal cells were investigated. PC 12 cells were exposed to THz radiation at beam incident power intensities of 0.25 W m(-2) (low intensity, LI), 0.5 W m(-2) (medium intensity, MI) and 1 W m(-2) (high intensity, HI) for 10 min. After exposure, the morphological and physiological status of the cells was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM imaging revealed that, after exposure to LI THz radiation, the cells exhibited membrane protrusions (blebs) measuring 70-120 nm in diameter. In contrast, cells exposed to HI THz radiation demonstrated increased uptake of FITC-dextran and nanospheres. Analysis of single-cell populations counterstained with 4 ' 6-diamidino-2-phenylindole (DAPI) showed a decrease in the proportion of DAPI-positive cells, with approximately 90, 80 and 50% remaining positive after exposure to LI, MI and HI THz radiation, respectively. However, only a slight increase in the proportion of dead cells was observed at varying THz intensities. Proteomic analysis of the cell changes following exposure to LI and HI THz irradiation indicated that THz radiation activated the CaN complex and upregulated genes involved in ribosome biogenesis and DNA damage repair.

Human primary T cells can be engineered for stable IL7 pathway activation. A, Schematic of CCR and sIL7, as well as transgenes engineered into cells. B, Transduction efficiency of T cells engineered to express GFP only, IL7R + GFP, or to secrete IL7 + GFP was measured by detecting eGFP positivity and CCR expression with flow cytometry. C, Concentration of IL7 measured in the supernatant of sIL7-transduced T cells and control cells (G-o) in cytokine-free culture medium. D, Immunophenotype characterization of T-cell subsets present in healthy donor and transduced T-cell preactivation (CD4 and CD8) and on day 8 of T-cell expansion [CCR and sIL7 treated with IL7: +IL7 (G-o); G-o]. E, Ratio of STAT5 phosphorylation to total STAT5 expression measured with TR-FRET in indicated cell types following 24 hours of cytokine starvation and following stimulation with IL7 for 30 minutes. Comparison between untreated and IL7-treated cells. F, Percent cells positive for CD127 expression measured by flow cytometry. Statistical comparison is between cell type and control (G-o, no treatment). G, Representative histograms from flow cytometric measurement of �127+ cells of each type. Data representative of n = 3 to 5 independent donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For DG, +IL7 indicates cells activated and expanded in the presence of IL7 prior to indicated analysis. H, MS proteomics analysis of IL7R signaling pathway downstream molecules for total protein and phosphosite abundance. Raw detected protein and phosphoprotein abundance were normalized to the control condition per donor and expressed as mean log2 fold change of three donors. *Note STAT5A pY694 and STAT5B pY699 are indistinguishable due to sequence identity. G-o, GFP-only.

Polyamines are small, polycationic molecules with amino groups that are present in most living organisms. Studies indicate that polyamines increase general protein synthesis and are essential for efficient translation. While progress has been made in understanding the role of polyamines in translation in bacteria and mammals, their contribution and mode of action in plants remain largely unexplored. In a previous study, we found that putrescine (Put) and the pathogen-associated molecular pattern (PAMP) from bacterial flagellin (flg22) transcriptionally induced ribosome biogenesis in plants. Here we examined the impact of polyamines (Put and spermine, Spm) and flg22 on ribosome complex formation in Arabidopsis. Our results indicate that polyamines, flg22 and their combinations increase the abundance of actively translating polysomes. Riboproteomic analyses revealed that polyamines and flg22 trigger differential changes in the accumulation of ribosomal proteins, which are structurally confined in response to Put. Importantly, Put was found binding to non-translating and actively translating ribosomes, suggesting that this polyamine has a role in functional aspects of translation, such as stabilization and/or remodeling of polysomal complexes. Additional global proteomics analyses in polyamine biosynthesis mutants revealed that lower Put availability triggers changes in proteins associated with ribonucleoprotein complex binding and biogenesis. Overall, our findings highlight the effect of polyamines and flg22 on shaping the ribosomal protein composition of actively translating ribosomes in plants.

This study aimed to identify protein markers for fibre type, and extent of post-mortem proteolysis, in bovine cutaneous trunci and masseter, which represent very different muscles types. We applied mass-spectrometry-based proteomic techniques to analysis of muscle fibre type and, examined temporal proteolysis changes with post-mortem ageing. Global proteomic analysis was performed on fresh beef masseter (high type-I fibres) and cutaneous trunci (high type-II fibres). The profile of semi-tryptic peptides, before and after ageing, was analysed to quantify protein degradation extent. Progression of proteolysis in type-I and type-II muscles was indicated by semi-tryptic peptides derived from heat-shock-protein 27, and troponin T fast skeletal muscle, respectively. The content of type I fibres in beef could be estimated using the abundance of myosin light chain-3, and enzymes involved in glycolysis could indicate the content of type II fibres. These markers could be used in the supply chain to monitor and improve beef quality.

Type 2 diabetes is a chronic metabolic disorder characterised by insulin resistance and sustained hyperglycaemia, and is a major cause of blindness, kidney failure, heart attacks and stroke. Our team has recently identified hexosaminidase A (HEXA) as an endocrine factor secreted by the liver that regulates sphingolipid metabolism in skeletal muscle. Specifically, HEXA converts GM2 to GM3 gangliosides within cell-surface lipid rafts. Remodelling of ganglioside composition by HEXA enhances IGF1 signalling in skeletal muscle, increasing muscle glucose uptake and improving blood glucose control. We produced a long-acting HEXA-FC fusion protein (murine HEXA and the fragment crystallisable [FC] region from IgG1) and evaluated the effects of chronic bi-weekly HEXA-FC administration (1 mg/kg body weight) on glycaemic control in C57BL/6 mice with diet-induced obesity and insulin resistance and the db/db mouse model of severe type 2 diabetes. Outcome measures included glucose and insulin tolerance, including a stable isotope-labelled GTT and assessment of tissue-specific glucose disposal, as well as proteomics analysis to define changes in skeletal muscle metabolism. Chronic administration of a long-acting recombinant HEXA-FC fusion protein led to improvements in random blood glucose, fasting blood glucose and glucose tolerance, driven by increased glucose disposal into skeletal muscle, effects that were associated with enhancement of IGF1 signalling in muscle. Given that skeletal muscle is a primary site of insulin resistance in individuals with type 2 diabetes, HEXA-FC protein therapy may open new avenues for therapeutic advancement in type 2 diabetes.

CD123-targeted CAR and CCR can be coexpressed with maintained functionality. A, Schematic showing CAR and CCR structures. B, Transduction efficiency measured using flow cytometric staining of Halo and SNAP tags. n = 3 to 6 unique T-cell donors, unless noted no significant difference between groups. C, Measurement of phosphorylated STAT5 percentage in cytokine starved, engineered T cells measured with and without activation on the immobilized rhCD123 target. n = 3 to 4 unique T-cell donors, comparison of CCR + CAR+ and CAR+ to NT (black) and with stimulation (colored). D, Soluble IL2 and IFNγ measured in the supernatant following coculture of unmodified (NT), CCR+, CAR+, or CCR + CAR+ T cells with CD123-negative (K562) and CD123-positive (K562.CD123, MV-4-11, and Molm-13) targets. n = 3 to 6 unique T-cell donors; data represented as mean ± SD. Significance noted is in comparison to NT (black asterisks) and/or CCR+ cells (green asterisks) or as noted. NT vs. CCR+ comparison was nonsignificant in all instances. E, Bioluminescence-based cytotoxicity assays performed using K562, K562.CD123, MV-4-11, and Molm-13 stably expressing ffLuc; n = 3 to 5 donors. For B–E, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Unless noted, comparisons were nonsignificant. NT, nontransduced.

The prominent bioactivities of fucoidan have attracted increasing attention from food scientists. Recent studies have highlighted the potential structural damages triggered by gastrointestinal digestion, which reduces the bioactive performance of fucoidan. Here, we aimed to preserve the bioactivities of fucoidan in a digestive environment by incorporating fucoidan at the interface of an oil/water emulsion. The effects of the digestive progression on fucoidan structures and bioactivities were traced by the dynamic changes in reducing sugar content and radical scavenging activities of digesta collected from different simulated digestion timepoints. Notably, there was a 71 % increase in the DPPH radical scavenging rate after fucoidan (0.25 %; w/w) was incorporated into an emulsion. Besides, the formulated emulsion showed remarkable storage stability at 4 °C with no cream layer formation for seven days. Although the acidified emulsion showed a 15.63 % creaming index, there was still a significant reduction in reducing sugar content after 2-h small intestinal digestion from 2.76 mg/mL for fucoidan digesta in a free form to 2.63 mg/mL for interfacial fucoidan digesta. Here, we highlighted that the fucoidan-stabilized emulsion retained superior antioxidant potential throughout the simulated gastrointestinal tract, which is possibly due to improved structural integrity. These data provide comprehensive information on the enhancement in physiochemical properties and bioactivities of fucoidan after transferring from a continuous phase to an interface, which suggests the potential application of fucoidan in functional fortified beverages and presents a novel supplementation approach to enhance its bioavailability in the health products

Abstract Background Nippostrongylus brasiliensis—a nematode of rodents—is commonly used as a model to study the immunobiology of parasitic nematodes. It is a member of the Strongylida—a large order of socioeconomically important parasitic nematodes of animals. Lipids are known to play essential roles in nematode biology, influencing cellular membranes, energy storage and/or signalling. Methods The present investigation provides a comprehensive, untargeted lipidomic analysis of four developmental stages/sexes (i.e. egg, L3, adult female and adult male stages) of N. brasiliensis utilising liquid chromatography coupled to mass spectrometry. Results We identified 464 lipid species representing 18 lipid classes and revealed distinct stage-specific changes in lipid composition throughout nematode development. Triacylglycerols (TGs) dominated the lipid profile in the egg stage, suggesting a key role for them in energy storage at this early developmental stage. As N. brasiliensis develops, there was a conspicuous transition toward membrane-associated lipids, including glycerophospholipids (e.g. PE and PC) and ether-linked lipids, particularly in adult stages, indicating a shift toward host adaptation and membrane stabilisation. Conclusions We provide a comprehensive insight into the lipid composition and abundance of key free-living and parasitic stages of N. brasiliensis. This study provides lipidomic resources to underpin the detailed exploration of lipid biology in this model parasitic nematode. Graphical Abstract

During acute oxidative phosphorylation (OXPHOS) dysfunction, reversal of succinate dehydrogenase (complex II) maintains the redox state of the Coenzyme Q (Q)-pool by using fumarate as terminal electron acceptor in certain tissues and cell lines. We identified the action of SDHAF2 protein, a complex II assembly factor, as critical for metabolic adaptation during complex III dysfunction in HEK293T cells. SDHAF2 loss during complex III inhibition led to a net reductive TCA cycle from loss of succinate oxidation, loss of SDHA active site-derived reactive oxygen species (ROS) signaling, insufficient glycolytic adaptation, and a severe growth impairment. Glycolysis adapted cells, however, did not accumulate SDHAF2 upon Q-pool stress, exhibited a net reductive TCA cycle and mild growth phenotypes regardless of SDHAF2 presence. Thus, our study reveals how complex II assembly controls a balance between dynamics of TCA cycle directionality, protection from Q-pool stress, and an ability to use ROS-meditated signaling to overcome acute OXPHOS dysfunction in cells reliant on mitochondrial respiration.