Biology subjects

Sanchez-Lopez, E.

Publications and source records attributed to Sanchez-Lopez, E..

3 recordsLinked to original sources

bioRxiv2023

Advancing multi-day ex vivo kidney perfusion using spatially resolved metabolomics

The ability to preserve metabolically active kidneys ex vivo for multiple days may permit reconditioning, repair and regeneration of deceased donor kidneys. However, the kidneys high metabolic demand limits its functional preservation. Current approaches focus on normothermic machine perfusion (NMP) at 37{degrees}C or hypothermic machine perfusion (HMP) at 4-8{degrees}C. At normothermia, kidneys are metabolically active but ex vivo preservation is limited to hours. During hypothermia kidneys can be preserved up to 24 hours but are metabolically inactive and suffer cold-induced injury. Therefore, we revisited sub normothermic perfusion (at 25{degrees}C) as an alternative approach to preserve human kidneys in a metabolically active state for extended periods of time. In a custom-made platform that includes a cell-free perfusate enriched with TCA cycle fuels, urine recirculation, and continuous hemofiltration we perfused discarded human kidneys up to 8 days. Using spatially resolved single cell resolution isotope tracing we demonstrate active metabolism in all the different renal cell types over this period. However, beyond 4 days cell composition of nephron segments assessed with spatial lipidomics changed substantially and injury markers such as NGAL and LDH increased in the perfusate. Up to 4 days, perfused human discarded donor kidneys maintained metabolic fluxes, functional parameters and allow for reperfusion using a porcine auto transplantation model. These data underpin that extended multi-day metabolic preservation of human kidneys is achievable using a sub normothermic perfusion platform.

bioengineering↗

bioRxiv2023

Novel determinant of antibiotic resistance: a clinically selected Staphylococcus aureus clpP mutant survives daptomycin treatment by reducing binding of the antibiotic and adapting a rod-shaped morphology

Daptomycin is a last-resort antibiotic used for treatment of infections caused by Gram-positive antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations, however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and {beta}-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that the improved survival of the clpP mutant strain during daptomycin treatment was associated with reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10 x MIC daptomycin resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression. IMPORTANCEThe bacterium Staphylococcus aureus is a leading cause of life-threatening infections and treatment is challenged by the worldwide dissemination of methicillin-resistant Staphylococcus aureus (MRSA) that are multi-drug resistant. Daptomycin, a cell membrane-targeting cationic lipopeptide, is one of the few antibiotics with activity against MRSA, however, the killing mechanism of daptomycin and the mechanisms leading to resistance are not fully understood. Here we show than an MRSA strain, isolated from the blood of a patient treated with daptomycin, has acquired a mutation that inactivates the ClpXP protease resulting in increased resistance to several antibiotics and diminished expression of virulence genes. Super resolution microscopy showed that the mutant avoids daptomycin-elicited killing by preventing the binding of the antibiotic to the septal site and by growing into a rod-shaped morphology. In summary, this study discloses new perspectives on the mechanism of killing and the mechanism of resistance to an antibiotic of last resort.

microbiology↗

bioRxiv2022

Single cell guided deconvolution of bulk transcriptomics recapitulates differentiation stages of acute myeloid leukemia and predicts drug response

The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power on drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stage (FAB classification) could be faithfully reconstituted using estimated cell compositions (ECCs). Moreover, we show that the ECCs reliably predict ex-vivo drug resistances as demonstrated for Venetoclax, a BCL-2 inhibitor, resistance specifically in AML with CD14+ monocyte phenotype. We further validate these predictions using in-house proteomics data by showing that BCL-2 protein abundance is split into two distinct clusters for NPM1-mutated AML at the extremes of CD14+ monocyte percentages, which could be crucial for the Venetoclax dosing for these patients. Our results suggest that Venetoclax resistance predictions can also be extended to AML without recurrent genetic abnormalities (NOS), and possibly to MDS-related AML and secondary AML. Collectively, we propose a framework for allowing a joint mutation and maturation stage modeling that could be used as a blueprint for testing sensitivity for new agents across the various subtypes of AML.

cancer biology↗