Biology subjects

Boulestreau, J.

Publications and source records attributed to Boulestreau, J..

4 recordsLinked to original sources

bioRxiv2025

Comparison of extracellular vesicles isolation methods reveals method-dependent protein and miRNA profiles in saliva

Salivary extracellular vesicles (EVs) represent a powerful, non-invasive source of biomarkers for disease diagnosis and monitoring. Their molecular cargo reflects systemic and local physiological states, offering a window into neurological and inflammatory disorders. However, the diversity of EV isolation protocols and the possibility that each enriches distinct EV subpopulations remains a major barrier to reproducibility and data comparability. We conducted a comprehensive comparison of three EV isolation methods: ultracentrifugation (UC), PEG-based precipitation (Q), and immunoaffinity capture (M) to evaluate their impact on EV yield, purity, and molecular composition. Salivary EVs from healthy volunteers were analysed using proteomic and small-RNA sequencing approaches. Principal component analysis revealed clear isolation method-dependent clustering, where M-derived EVs displayed the most distinct profile. UC and Q produced broader proteomic repertoires with higher total protein content, whereas M-isolated EVs exhibited greater purity and enrichment of trafficking-and lysosome-associated proteins. Over 731 miRNAs selected, 28 were consistently altered across methods and 65 uniquely enriched in M isolates. RT-qPCR confirmed key directional trends. These 93 method-dependent miRNAs have predicted targets associated with synaptic structure and neurodegenerative pathways. These findings show that isolation methodology deeply shapes salivary EV cargo and suggest that immunoaffinity capture can isolate specific EV populations meeting diagnostic requirements.

molecular biology↗

bioRxiv2025

Capturing in saliva real time emotions induced by a fragrance: an objective emotional assessment based on multiplex molecular biomarker profiles

This study introduces a non-invasive approach to objectively assess fragrance-induced emotions using multiplex salivary biomarker profiling. Traditional methods such as self-reports, physiological monitoring, or neuroimaging are often limited by subjectivity, invasiveness, or poor temporal resolution. Saliva offers a practical alternative, reflecting rapid neuroendocrine changes linked to emotional states. We analyzed four key salivary biomarkers: cortisol (stress, HPA-axis activity), alpha-amylase (sympathetic activation), dehydroepiandrosterone (resilience), and oxytocin (social bonding, emotional regulation) to capture multidimensional emotional responses. Two clinical studies (N=30, N=63) and one consumer study (N=80) exposed healthy volunteers to six fragrances, with saliva collected before, 5 minutes after, and 20 minutes after olfactory stimulation. Subjective ratings of happiness, relaxation, confidence, and dynamism were also obtained via questionnaires. Rigorous analytical validation accounted for reproducibility, circadian variation and sample stability. Biomarker patterns revealed fragrance-specific emotional profiles, with distinct subgroups of participants whose biomarker dynamics correlated with specific emotional states. Increased oxytocin and decreased cortisol consistently aligned with happiness and relaxation, whereas distinct biomarker combinations predicted confidence or dynamism. Classification and regression tree analysis demonstrated high sensitivity for detecting these profiles. Validation in an independent cohort (N=80) using an implicit association test confirmed concordance between molecular profiles and behavioral measures, underscoring the robustness of this method. These findings establish salivary biomarker profiling as a reliable tool for decoding real-time emotional responses. Beyond scientific insights into affective neuroscience, this approach holds translational potential in personalized fragrance design, sensory marketing, and therapeutic applications for stress-related disorders. Expanding the biomarker panel and integrating molecular data with neuroimaging or autonomic measures could further elucidate the interplay between central olfactory processing and peripheral physiology.

neuroscience↗

bioRxiv2024

Assessment of salivary microRNA by RT-qPCR: Challenges in data interpretation for clinical diagnosis

Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. MethodsSaliva was sampled from ten healthy volunteers for a time course analysis. A panel of six miRNA targets (with different sequence homologies) were analysed by one of the most commonly used commercially available RT-qPCR kit. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs. The reliability of all tested assays to differentiate miRNA expression profiles were analysed, to statistically discriminate background noise from intrinsic individual signals. ResultsSignificant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2-4 logs higher than that of the limit of detection (LOD), the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. ConclusionsOur results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. In particular, assessment of fundamental parameters including LOD, LOQ and crosstalk of each assay is strictly necessary to interpret observed variations. The standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies. However, further technological breakthroughs are still required to overcome discrepancies in order to accelerate the translation of salivary miRNAs for clinical applications.

molecular biology↗

bioRxiv2024

Salivary extracellular vesicles isolation methods impact the robustness of downstream biomarkers detection

Extracellular vesicles (EVs), crucial mediators in cell-to-cell communication, are implicated in both homeostatic and pathological processes. Their detectability in easily accessible peripheral fluids like saliva positions them as promising candidates for non-invasive biomarker discovery. However, the lack of standardized methods for salivary EVs isolation greatly limits our ability to study them. Therefore, we rigourously compared salivary EVs isolated using two scalable techniques--co-precipitation and immuno-affinity--against the long-established but labor-intensive ultracentrifugation method. Employing Cryo-Electron Microscopy, Nanoparticle Tracking Analysis, Western blots (WB), and proteomics, we identified significant method-dependent variances in the size, concentration, and protein content of EVs. Importantly, our study uniquely demonstrates the ability of EV isolation to detect specific biomarkers that remain undetected in whole saliva by WB. RT-qPCR analysis targeting six miRNAs confirmed a consistent enrichment of these miRNAs in EV-derived cargo across all three isolation methods. We also found that pre-filtering saliva samples with 0.22 or 0.45 {micro}m pores adversely affects subsequent analyses. Our findings highlight the untapped potential of salivary EVs in diagnostics and advocate for the co-precipitation method as an efficient, cost-effective, and clinically relevant approach for small-volume saliva samples. This work not only sheds light on a neglected source of EVs but also paves the way for their application in routine clinical diagnostics.

molecular biology↗