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Introduction: Pregnant women in sub-Saharan Africa face heightened susceptibility to Plasmodium falciparum malaria, with placental sequestration driving adverse outcomes. The infection may lead to pregnancy-associated malaria (PAM) because of the sequestration of Plasmodium falciparum-infected erythrocytes in the placental intervillous space. Although there are several tools for diagnosing malaria infection during pregnancy, including blood smear microscopic examination, rapid diagnostic tests, and PCR, there are no tools for detecting placental infection and, by extension, any dysfunction associated with PAM. Thus, PAM, specifically placental infection, can only be confirmed via postnatal placental histopathology. Therefore, there is an urgent need for specific plasma biomarkers of PAM.

Methods: Here, we used the high throughput proximity extension assay to screen plasma from malaria-exposed pregnant women for differentially expressed proteins that may serve as candidate biomarkers of Plasmodium falciparum infection during pregnancy, with future potential to inform diagnosis of PAM or adverse malaria outcomes. Such biomarkers may also elucidate the pathophysiology of PAM.

Results: Using proximity extension assay (PEA), we identified elevated IgG Fc receptor IIb (FCGR2B) and heme oxygenase-1 (HO-1) in malaria-positive pregnancies, while neurturin (NRTN) and IL-20 were downregulated.

Discussion: IL-20 emerged as a top candidate biomarker, warranting validation in large cohorts with placental histopathology.

Background

Gastric cancer (GC), the world’s fifth most common malignancy, remains a major cause of death, motivating minimally invasive protein measurements. Small extracellular vesicles (sEVs) mirror cellular state but are difficult to profile from limited material. Proximity extension assay (PEA) enables multiplex, sensitive detection. We developed a PEA-centred workflow to profile GC cell-line sEVs, benchmarked against cells and an EV-rich control, and nominate hypothesis-generating candidates.

Methodss

EVs from four GC cell lines (Kato III, AGS, MKN45, MKN7) were isolated by differential centrifugation/filtration and ultracentrifugation; prostasomes (seminal-fluid EVs; ethics-approved) were purified by ultracentrifugation, Superdex 200 size-exclusion, and sucrose gradient. EVs were characterized by TEM, NTA, and Western blot (CD9/CD63/CD81; Calnexin). Protein profiling used Olink PEA across five Target 96 panels, two commercial (Oncology, Inflammation) and three experimental (Cancer, Cellular Pathways, Neurology) with availability-based selection; single replicate per condition. NPX values were background/LOD-corrected in R, with PCA. STRING PPI used medium confidence (0.4) with FDR-adjusted enrichment. Selected proteins were validated by SP-PLA (qPCR), EV-PLA (flow cytometry), and WB.

Resultss

EVs and prostasomes were purified and characterized per MISEV2023 (TEM/NTA; CD9/CD63/CD81+, calnexin–). Using five Olink PEA panels, 460 proteins were profiled; principal component analysis separated sEVs from parental cells. We identified 71 proteins enriched in GC sEVs, including EphA2, cMet, IL-6, SCF, AXIN1, NOTCH1, and CAV1; 17 were unique to Kato III sEVs. STRING showed a highly connected network, with top pathways: Pathways in cancer, Cytokine–cytokine receptor interaction, and PI3K–Akt. Orthogonal assays (SP-PLA, EV-PLA, WB densitometry) confirmed the presence of EphA2 and cMet on GC sEVs.

Discussions

PEA-based profiling of GC cell-line sEVs nominates 71 enriched proteins (e.g., EphA2, cMet, NOTCH1, CAV1) as hypothesis-generating candidates that may reflect cell-type–specific cargo and tumor–microenvironment signaling. Key limitations include the in-vitro design, single replicates, preselected PEA panels, and the use of prostasomes rather than healthy plasma sEVs, which may affect generalizability and specificity. Translational relevance requires validation in patient biofluids with orthogonal assays and cohort-level evaluation.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues.

This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs.

Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.