Biology subjects

Molina, F.

Publications and source records attributed to Molina, F..

10 recordsLinked to original sources

bioRxiv2026

The Glycoproteome of Loxosceles Venom Reveals an Expanded Landscape of Animal Toxins

Spider venoms of the genus Loxosceles constitute a complex mixture of bioactive proteins and peptides associated with the major clinical manifestations of loxoscelism. Although significant advances have been made in the proteomic characterization of these toxins, studies addressing post-translational modifications, particularly glycosylation, and their potential impact on venom properties remain scarce. Here, we investigated the occurrence and characteristics of N-glycosylation in the venoms of Loxosceles gaucho, Loxosceles intermedia, and Loxosceles laeta. Using two-dimensional electrophoresis combined with glycoprotein-specific staining and LC-MS/MS, we identified glycosylation in different proteins biologically relevant to envenomation, predominantly belonging to the phospholipase D family, with distinct profiles among the species analyzed. Bottom-up glycoproteomic analysis of crude venoms using Byonic and a customized sequence database identified 88 N-glycosylated peptide sequences, primarily associated with lyases and hydrolases. Extensive microheterogeneity was detected at individual glycosylation sites, with paucimannosidic structures predominating alongside oligomannose and hybrid/complex N-glycans. Complementary enzyme-linked lectin assays supported the presence of distinct carbohydrate epitopes, including mannose, galactose, and N-acetylgalactosamine containing structures, and revealed 2,6-sialic acid-reactive motifs in two species. These findings establish N-glycosylation as an additional layer of molecular heterogeneity in Loxosceles venoms and provide a foundation for investigating its potential contribution to toxin properties and venom diversity.

biochemistry↗

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

Coevolution and temporal dynamics of species interactions shape species coexistence

Previous research has revealed how species traits determine species interactions, and how species interactions influence species coexistence. However, this hierarchical view ignores the coevolutionary feedback from species interactions to species traits and its consequences for species coexistence. Here, we developed a theoretical model of quantitative genetics to explore how the coevolution of morphological and phenological traits shapes the structure and stability of mutualistic interaction networks. We found that, in comparison to morphological traits, phenological traits led to distinctive species evolutionary trajectories, resulting in different emergent properties at community level. This is because phenological traits promoted facilitation over competition. While morphological coevolution gave rise to modular and specialized interaction networks with a negative diversity-stability trade-off, phenological coevolution was required for the emergence of nested interaction networks that exhibited a positive relationship between diversity and structural stability. Empirical observations from 17 pollination networks were consistent with the theoretical results: we found many phenological motifs promoting facilitation over competition, suggesting an important role of phenological coevolution in community assembly. The seasonal organization of empirical interactions enhanced the community stability and dampened the diversity-stability trade-off that was observed when aggregating interactions across time. Our results highlight the importance of phenological coevolution in the emergence of diverse and stable communities.

ecology↗

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↗

bioRxiv2021

Living to the high extreme: unraveling the composition, structure, and functional insights of bacterial communities thriving in the arsenic-rich Salar de Huasco - Altiplanic ecosystem.

Microbial communities inhabiting extreme environments like Salar de Huasco (SH) are adapted to thrive while exposed to several abiotic pressures and the presence of toxic elements like arsenic (As). Hence, we aimed to uncover the role of arsenic in shaping bacterial composition, structure, and functional potential in five different sites in this Altiplanic wetland using a shotgun metagenomic approach. The sites exhibit wide gradients of arsenic (9 to 321 mg/kg), and our results showed highly diverse communities and a clear dominance exerted by the Proteobacteria and Bacteroidetes phyla. Functional potential analyses showed broadly convergent patterns, contrasting with their great taxonomic variability. Arsenic-related metabolism is different among the five communities, as well as other functional categories like those related to the CH4 and S cycles. Particularly, we found that the distribution and abundance of As-related genes increase, following along the As concentration gradient. Approximately 75% of the detected genes for As-metabolism belong to expulsion mechanisms, being arsJ and arsP pumps related to sites with higher As concentrations and present almost exclusively in Proteobacteria. Furthermore, taxonomic diversity and functional potential are reflected in the 12 reconstructed high-quality MAGs (Metagenome Assembled Genomes) belonging to the Bacteroidetes (5), Proteobacteria (5), Cyanobacteria (1) and Gemmatimonadota (1) phyla. We conclude that SH microbial communities are diverse and possess a broad genetic repertoire to thrive under extreme conditions, including increasing concentrations of the highly toxic As. Finally, this environment represents a reservoir of unknown and undescribed microorganisms, with a great metabolic versatility, which needs further study. IMPORTANCEMicrobial communities inhabiting extreme environments are fundamental for maintaining the ecosystems; however, little is known about their potential functions and interactions among them. We sampled the microbial communities in Salar de Huasco (SH) in the Chilean Altiplano, a fragile and complex environment that comprises several stresses. We found that microbes in SH are taxonomically diverse; nonetheless, their functional potential seems to have an important convergence degree, suggesting high adaptation levels. Particularly, arsenic metabolism showed differences associated with increasing concentrations of the metalloid throughout the area, and it is effectively exerting a clear and significant pressure over these organisms. Thus, this researchs significance is that we described highly specialized communities thriving in little-explored environments under several pressures, considered analogous of early Earth and other planets, and can have the potential for unraveling technologies to face climate change repercussions in many areas of interest.

microbiology↗

bioRxiv2018

PEPOP: new approaches to mimic non-continous epitopes

Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits as replacing a protein difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitope predicted by bioinformatics tools are commonly used and these sequential epitopes are used as such in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools proposed so far to predict peptide sequences: Superficial and PEPOP. PEPOP can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. We have developed an improved version of PEPOP dedicated to answer to the experimentalists need for a tool able to handle proteins and to turn them into peptides. The PEPOP web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature.\n\nWe present the user-friendly, well-structured web-site of PEPOP and its validation through the use of predicted immunogenic or antigenic peptides mimicking discontinuous epitopes in different experimental ways. PEPOP proposes 35 methods of peptide design to guide experimentalists in using peptides potentially capable of replacing the cognate protein in its interaction with an Ab.

bioinformatics↗

bioRxiv2018

Benchmarking the PEPOP methods for mimicking discontinuous epitopes

Computational methods provide approaches to identify epitopes in protein antigens to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein antigen in its interaction with an antibody. In the present work, we describe these new methods and the benchmarking of their performances.\n\nBenchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 antigen-antibody complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method.\n\nThe PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, \"optimized\" methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.

bioinformatics↗