Biology subjects

Kahli, M.

Publications and source records attributed to Kahli, M..

7 recordsLinked to original sources

bioRxiv2026

miRNova: A Next-Generation Platform for Ultra-Precise and Highly Specific MicroRNA Quantification Integrating a Tailored Stem Loop RT-qPCR and a Robust Analytical Framework

MicroRNAs (miRNAs) are ultra-short RNA molecules characterized by high sequence homology, frequent post-transcriptional modifications, and typically low abundance, particularly in circulating biofluids. These inherent biological features present substantial technical challenges for RT-qPCR- based quantification. Consequently, the development of miRNA RT-qPCR assays has required architectural adaptations at the reverse transcription (RT) stage to generate extended cDNA templates, thereby enabling effective downstream quantitative PCR amplification. One widely adopted approach involves the enzymatic addition of a poly(A) tail to the 3' end of miRNAs, followed by poly(T)-primed universal reverse transcription, which has gained broad acceptance due to its perceived sensitivity and simplified workflow. However, independent experimental evidence indicates that this architecture does not consistently provide the level of specificity required for reliable single-nucleotide (SN) discrimination, particularly when quantifying low-abundance circulating miRNA targets, as demonstrated in our previous study. An alternative strategy relies on miRNA-specific reverse transcription using stem-loop priming has been equally well accepted. When generically generated, this approach offers certain improved specificity, but its performance in resolving single-nucleotide differences remains limited. In this article, we employed precision engineering to maximize specificity for both reverse transcription and qPCR steps. By tailoring both primer design and reaction architecture to the specific sequence features of each miRNA, we enable robust single nucleotide discrimination among these ultra-short targets. Prototype of ten different miRNova assays quantifying miRNAs whose sequences are differed in various configurations were tested on synthetic miRNA targets. For miRNova assay validation, saliva samples were elite rugby players submitted to small RNA extraction, then RT-qPCR. Spike-in of synthetic targets was applied for each quantification point to characterized the sensitivity, specificity and accuracy of the assays. Comparative analysis was performed between miRNova and two commercially available kits on the same sample set. The obtained results show a superior performance of miRNova assays allowing for sensitive and accurate quantification of miRNAs in saliva samples. Altogether, this results in modular, reproducible assays optimized for low-abundance miRNA detection in challenging biofluids, including saliva, positioning the platform beyond existing sensitivity-focused solutions toward true diagnostic-grade specificity.

bioengineering↗

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↗

bioRxiv2018

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5 flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5 flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

molecular biology↗

bioRxiv2018

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

molecular biology↗