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Ciordia, S.

Publications and source records attributed to Ciordia, S..

8 recordsLinked to original sources
bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗
bioRxiv2025

Multi-omics integrative approach of senescent endothelial cells and derived extracellular vesicles in a replicative senescence model

Aging is a major unmodifiable risk factor for cardiovascular disease (CVD). Replicative endothelial senescence (RES), characterized by permanent cell-cycle arrest and a senescence-associated secretory phenotype (SASP), is a hallmark of vascular aging. However, the molecular mechanisms underlying RES, particularly the role of extracellular vesicles (EVs), remain poorly understood. To this aim, an integrated multi-omics approach (proteomics, mRNA, and miRNA profiling) was applied to characterize senescent and early-passage human umbilical vein endothelial cells (HUVECs) and their secreted EVs. Senescent HUVECs and EVs displayed a canonical senescence profile, including cell-cycle arrest, DDR activation, NF-{kappa}B signaling, and SASP induction, alongside marked suppression of RNA metabolism, ribosome biogenesis, and DNA repair. Multi-omics integration has linked endothelial senescence to vascular aging, maladaptive angiogenesis, and ECM remodeling, which are key processes in CVD development. Moreover, senescent EVs propagate senescence by exporting fewer reparative and antioxidant factors while carrying pro-fibrotic, adipogenic, and angiogenic mediators. Multi-omics profiling revealed consistent transcript-protein changes in senescent cells and EVs, defining a robust molecular signature of RES, including endothelial cell-specific markers and post-transcriptional regulators (lncRNAs and miRNAs). Among these, miR-22-3p and miR-126-5p emerged as key modulators in both cells and EVs, with miR-22-3p demonstrating compartment-specific regulation. Integration further uncovered a coordinated regulatory network involving miRNAs (miR-23, miR-335-3p, miR-29 family, miR-590-3p, and miR-126-5p) that were inversely correlated with transcription factors (e.g., REST, KLFs, and ZNFs) and downstream targets, collectively driving endothelial senescence through impaired PI3K-Akt signaling. Although further validation is needed, these findings provide new insights into the molecular mechanisms of RES and open perspectives for modulating secondary senescence and age-related vascular diseases.

cell biology↗
bioRxiv2024

Time-based quantitative proteomic and phosphoproteomic analysis of A549-ACE2 cells during SARS-CoV-2 infection

The outbreak of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2, led to an ongoing pandemic with devastating consequences for the global economy and human health. With the global spread of SARS-CoV-2, multidisciplinary initiatives were launched to explore new diagnostic, therapeutic, and vaccination strategies. From this perspective, proteomics could help to understand the mechanisms associated with SARS-CoV-2 infection and to identify new therapeutic targets for antiviral drug repurposing and/or discovery. A TMT-based quantitative proteomics and phosphoproteomics analysis was performed to study the proteome remodeling of human lung alveolar cells transduced to express human ACE2 (A549-ACE2) after infection with SARS-CoV-2. Targeted PRM analysis was performed to assess the detectability in serum and prognostic value of selected proteins. A total of 6802 proteins and 6428 phospho-sites were identified in A549-ACE2 cells after infection with SARS-CoV-2. Regarding the viral proteome, 8 proteins were differentially expressed after 6 h of infection and reached a steady state after 9 h. In addition, we detected several phosphorylation sites of SARS-CoV-2 proteins, including two novel phosphorylation events at S410 and S416 of the viral nucleoprotein. ImportanceThe differential proteins here identified revealed that A549-ACE2 cells undergo a time-dependent regulation of essential processes, delineating the precise intervention of the cellular machinery by the viral proteins. From this mechanistic background and by applying machine learning modelling, 29 differential proteins were selected and detected in the serum of COVID-19 patients, 14 of which showed promising prognostic capacity. Targeting these proteins and the protein kinases responsible for the reported phosphorylation changes may provide efficient alternative strategies for the clinical management of COVID-19.

biochemistry↗
bioRxiv2023

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial Intercellular Adhesion Molecule (ICAM)-1 is apically polarized, interacts with and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures (BCs). We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

cell biology↗
bioRxiv2023

Molecular basis of Progressive Familial Intrahepatic Cholestasis 3. A proteomics study.

Background and aimsProgressive familiar intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease which affects between 1/50,000 to 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids are kept into phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free bile acids that results in liver damage. PFIC3 onset occurs usually at early ages, progress rapidly and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short-aged patients. MethodsTo gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. ResultsWe identified 6,246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5,090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprograming, cytoskeleton and extracellular matrix remodeling and cell proliferation were identified as main drivers of the disease. ConclusionOur results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients. Lay Summary/Key PointsPFIC3 is a rare disease that affect 1/50,000 to 1/100,000 children that present severe symptoms in the first years of life and have scarce therapeutic options. We identified a panel of proteins that recapitulate some of the main cellular processes associated to the progression of liver injury in PFIC3 patients and suggest alternative treatment options.

molecular biology↗
bioRxiv2021

An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

A revised model of the aromatic binding A domain of the {sigma}54-dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR, and DmpR. The resulting frame was instrumental for mapping the large number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic-binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc-binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped. Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain --let alone the whole-length protein-- has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.

microbiology↗
bioRxiv2019

Differential polysaccharide utilization is the basis for a nanohaloarchaeon : haloarchaeon symbiosis.

Nanohaloarchaeota, a clade of diminutive archaea, with small genomes and limited metabolic capabilities, are ubiquitous in hypersaline habitats, which they share with the extremely halophilic and phylogenetically distant euryarchaea. Some of these nanohaloarchaeota and euryarchaea appear to interact with each other. In this study, we investigate the genetic and physiological nature of their relationship. We isolated the nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh and the haloarchaeon Halomicrobium sp. LC1Hm from a solar saltern, reproducibly co-cultivated these species, sequenced their genomes, and characterized their metabolic/trophic interactions. The nanohaloarchaeon is a magnesium-dependent aerotolerant heterotrophic anaerobe of the DPANN superphylum; it lacks respiratory complexes and its energy production relies on fermentative metabolism of sugar derivatives, obtained by depolymerizing alpha-glucans or by acquiring the chitin monomer N-acetylglucosamine from the chitinolytic haloarchaeal host. Halomicrobium is a member of the class Halobacteria and a chitinotrophic aerobe. The nanohaloarchaeon lacks key biosynthetic pathways and is likely to be provided with amino acids, lipids, nucleotides and cofactors via physical contact with its host Halomicrobium. In turn, the ability of Ca. Nanohalobium to hydrolyse alpha-glucans boosts the hosts growth in the absence of a chitin substrate. These findings suggest that at least some members of the nanohaloarchaea, previously considered ecologically unimportant given their limited metabolic potential, in fact may play significant roles in the microbial carbon turnover, food chains, and ecosystem function. The behaviour of Halomicrobium, which accommodates the colonization by Ca. Nanohalobium, can be interpreted as a bet-hedging strategy, maximizing its long-term fitness in a habitat where the availability of carbon substrates can vary both spatially and temporarily.

microbiology↗