Biology subjects

Delcea, M.

Publications and source records attributed to Delcea, M..

4 recordsLinked to original sources

bioRxiv2026

Exosome-mediated LNA anti-miR delivery achieves potent miR-21 inhibition and PTEN restoration in podocytes.

BackgroundPodocyte injury and loss are central drivers of chronic kidney disease (CKD) progression and are involved in the majority of glomerular diseases. Among the molecular pathways implicated in podocyte dysfunction, microRNA-21 (miR-21) is consistently upregulated during glomerular injury and suppresses protective target genes including PTEN. Although anti-miR-21 approaches have shown high efficacy in preclinical models for Alport Syndrome and diabetic nephropathy, clinical translation of free anti-miR-21 oligonucleotides remains challenging, potentially reflecting limited glomerular and podocyte target engagement. Here, we show that an exosome-mediated delivery of an LNA-miR-21 inhibitor results in a higher intracellular uptake, leading to functional miR-21 inhibition and PTEN restoration in podocytes. MethodsExosomes were isolated from immortalized murine podocytes SVI and were directly loaded with fluorescently-labeled LNA anti-miR-21 (LNA-21) and anti-miR-Ctrl. Exosome integrity was assessed by transmission electron microscopy, dynamic light scattering and Western blot analysis of CD9 and TSG101. Uptake efficiency and intracellular delivery were analyzed by confocal laser-scanning microscopy, time-series imaging (2-48 h) and (imaging) flow cytometry. Functional efficacy was evaluated by TaqMan RT-qPCR and PTEN protein expression in differentiated SVI podocytes, primary murine podocytes and puromycin aminonucleoside (PAN)-injured primary glomeruli. ResultsExosome number, morphology and marker expression remained unchanged after direct LNA-miR-21 loading. Time-series imaging demonstrated progressive intracellular accumulation of exosome-delivered LNA-21 cargo from 2 to 48 h, whereas free LNA-21 showed no detectable uptake. Flow cytometry and imaging flow cytometry confirmed an efficient (76.6%) intracellular delivery exclusively via exosomes. Exosome-mediated LNA-21 delivery induced a robust miR-21 suppression, resulting in an 99% reduction in immortalized podocytes, a 97% reduction in primary podocytes and a 97% reduction in PAN-injured glomeruli (all p < 0.05). In contrast, free LNA-21 did not achieve significant suppression in all investigated models. To evaluate the functional consequences of miR-21 inhibition, we examined PTEN expression, as PTEN is a known direct target of miR-21. Notably, restoration of PTEN protein expression was observed only following exosome-mediated LNA delivery which was confirmed by Western blot and immunofluorescence, suggesting efficient functional inhibition of miR-21 in podocytes. Transfection of scrambled controls confirmed sequence specificity throughout. ConclusionThese findings demonstrate that exosome-mediated LNA delivery overcomes the limited uptake of free inhibitor oligonucleotides in podocytes and enables functional inhibition of miR-21 across multiple experimental systems. These data provide mechanistic proof-of-principle for exosome-based miR inhibitor delivery as a potential strategy to improve podocyte-targeted therapies in CKD.

cell biology↗

bioRxiv2025

Wrapping it Up: Structural Basis of ADAMTS13 Global Latency

ADAMTS13 is a critical enzyme responsible for cleaving ultra-large von Willebrand factor (VWF) multimers, thereby preventing the formation of microthrombi in the microvasculature. Dysfunction or deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura (TTP), a rare and life-threatening disorder characterized by microangiopathic hemolytic anemia and severe thrombocytopenia. Understanding the regulation of ADAMTS13 is essential for developing therapeutic interventions for TTP. ADAMTS13 exhibits two latency mechanisms: local latency within the metalloprotease (MP) domain and global latency that affects its overall conformation. Although the role of the two CUB domains in binding the Spacer module is known during global latency, the contributions of TSP7, TSP8, and the flexible Linker region (L3) between TSP8 and CUB1 have been repeatedly observed but never explained. Binding studies with monoclonal antibodies (mAbs) targeting the MP domain have revealed a cryptic epitope that becomes accessible only when ADAMTS13 is conformationally activated, and Spacer-CUB binding is disrupted. However, the mechanism behind this long-range effect has never been explained. We present a novel autoinhibition model of ADAMTS13, proposing that its distal domains directly occlude substrate binding sites, thereby modulating the enzymes activity. In this model, TSP7 and TSP8 bind directly to the MP module, while the Linker region between TSP8 and CUB1 acts as a pseudosubstrate, mimicking the natural VWF-A2 substrate and blocking the binding sites. Our results show that the CUB domains must depart from the tandem arrangement observed in the crystal structure and instead adopt refined binding positions. Further evidence comes from novel monoclonal antibodies targeting more cryptic epitopes on various ADAMTS13 domains that we have recently presented, as well as pH- and EDTA-dependent conformational changes observed by ELISA. A detailed molecular model is derived from extensive molecular simulations and mechanistically reconciles a wide range of previously disconnected experimental observations. This novel insight into the regulation of ADAMTS13 lays the groundwork for new therapeutic strategies against TTP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/689657v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@abed60org.highwire.dtl.DTLVardef@16d7470org.highwire.dtl.DTLVardef@f7a14aorg.highwire.dtl.DTLVardef@17e350f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAtomistic Structure of autoinhibited ADAMTS13-Del3To6 C_LIO_LIL3 linker (TSP8-CUB1) has a dual function: acting as a pseudosubstrate that blocks VWF-A2 binding sites and guiding CUB1-2 positioning C_LIO_LITSP7 and TSP8 bind at the MP domain to restrain activity C_LIO_LICUB1 and CUB2 domains are not a single functional block C_LI

molecular biology↗

bioRxiv2024

SusC/D-like proteins in Gammaproteobacteria that utilize fructans

Fructans are ubiquitous in terrestrial ecosystems, however, these glycans are unexplored in the marine environment. We have discovered that the Antarctic gammaproteobacterium Pseudoalteromonas distincta is highly adapted to the degradation of fructose-containing substrates. This is enabled by proteins encoded in several genomic regions, including a fructan polysaccharide utilization locus (PUL). In addition to a glycoside hydrolase from family 32 (GH32), the fructan PUL encodes two proteins that have been described as specific for Bacteroidota and were previously unknown for Gammaproteobacteria: a glycan-binding SusD-like protein and a SusC-like TonB-dependent transporter (TBDT), which work as a complex in glycan import. Proteome analyses and biochemistry results suggest that the SusC/D-like proteins of P. distincta shuttle small-sized inulin-type fructans directly into the cell, where they are degraded by a periplasmic exo-active GH32. A SusD-like protein could provide a competitive adavantage in the absence of extracelluar endo-active inulinases. Comparative genomics identified further SusC/D-like proteins in Gammaproteobacteria, most of which are co-encoded with GH32s, indicative of fructan PULs, and are frequently associated with the marine habitat. Our study thus shows the first known exception to the paradigm that only Bacteroidota use SusC/D-like proteins. It further suggests that fructans contribute to the marine glycan pool.

molecular biology↗

bioRxiv2022

The inhibitory effect of EGCG-AgNP-lysozyme bioconjugates on biofilm formation

Biofilms are multicellular communities of microbial cells that grow on natural and synthetic surfaces. They have become the major cause for hospital-acquired infections because once they form, they are very difficult to eradicate. Nanotechnology offers a new approach to fight biofilm-associated infections. Here, we report on the synthesis of silver nanoparticles (AgNPs) with antibacterial ligand epigallocatechin gallate (EGCG) and the formation of lysozyme protein corona on AgNPs as shown by UV-Vis, dynamic light scattering, and circular dichroism analyses. We further tested the activity of EGCG-AgNPs and their lysozyme bioconjugates on the viability of Bacillus subtilis cells and biofilm formation. Our results showed that, although EGCG-AgNPs presented no antibacterial activity on planktonic Bacillus subtilis cells, they inhibited B. subtilis biofilm formation at concentrations larger than 40 nM and EGCG-AgNP-lysozyme bioconjugates inhibited biofilms at concentrations above 80 nM. Cytotoxicity assays performed with human cells showed a reverse trend, where EGCG-AgNPs barely affected human cell viability, while EGCG-AgNP-lysozyme bioconjugates severely hampered viability. Our results therefore demonstrate that EGCG-AgNPs may be used as non-cytotoxic antibiofilm agents.

biophysics↗