by Keyword: Plasmids
Fraire JC, Prado-Morales C, Aldaz Sagredo A, Caelles AG, Lezcano F, Peetroons X, Bakenecker AC, Di Carlo V, Sánchez S, (2024). Swarms of Enzymatic Nanobots for Efficient Gene Delivery Acs Applied Materials & Interfaces 16, 47192-47205 
This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.
JTD Keywords: Animals, Barrier, Cell line, tumor, Dna, Drug delivery, Drug-delivery, Enzyme catalysis, Gene delivery, Gene transfer techniques, Humans, Lactic acid, Mice, Nanobots, Nanoparticles, Pdna, Plasmids, Polyglycolic acid, Polylactic acid-polyglycolic acid copolymer, Swarming, Transfectio, Transfection, Urease, Urinary bladder neoplasms
Hüttener, M, Hergueta, J, Bernabeu, M, Prieto, A, Aznar, S, Merino, S, Tomás, J, Juárez, A, (2022). Roles of Proteins Containing Immunoglobulin-Like Domains in the Conjugation of Bacterial Plasmids Msphere 7, e00978-21
Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.
JTD Keywords: antimicrobial resistance, bacterial ig-like proteins, bacterial lg-like proteins, chromosomal genes, identification, inca/c, mutational analysis, plasmid conjugation, products, r-factors, resistance plasmids, salmonella-enterica, sequence, Antimicrobial resistance, Bacterial ig-like proteins, Escherichia-coli, Plasmid conjugation
Seuma, M, Faure, AJ, Badia, M, Lehner, B, Bolognesi, B, (2021). The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer's disease mutations Elife 10, e63364
Plaques of the amyloid beta (A beta) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in A beta also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of A beta. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in A beta, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in A beta.
JTD Keywords: aggregation, kinetics, oligomers, onset, rates, state, Aggregation, Alzheimer disease, Alzheimer's, Amyloid, Amyloid beta-peptides, Computational biology, Deep mutagenesis, Dna mutational analysis, Genetics, Genomics, High-throughput nucleotide sequencing, Kinetics, Mutation, Nucleation, Oligomers, Onset, Plasmids, Precursor protein, Rates, S. cerevisiae, Saccharomyces cerevisiae, State, Systems biology
Tassinari, E., Aznar, S., Urcola, I., Prieto, A., Hüttener, M., Juárez, A., (2016). The incC sequence is required for R27 plasmid stability Frontiers in Microbiology 7, (6), Article 629
IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.
JTD Keywords: Antimicrobial resistance, E protein, IncC, IncHI1 plasmids, Plasmid R27, Plasmid stability
