Biology subjects

Tomas-Gallardo, L.

Publications and source records attributed to Tomas-Gallardo, L..

3 recordsLinked to original sources

bioRxiv2024

Drug screening targeting TREM2-TYROBP transmembrane binding

TREM2 encodes a microglial membrane receptor involved in the disease-associated microglia (DAM) phenotype whose activation requires the transmembrane interaction with TYROBP. Mutations in TREM2 represent a high-impact risk factor for Alzheimers disease (AD) which turned TREM2 into a significant drug target. We present a bacterial two-hybrid (B2H) system designed for high-throughput screening of modulators for the TREM2-TYROBP transmembrane interaction. In a pilot study, 315 FDA-approved drugs were analyzed to identify potential binding modifiers. Our pipeline includes multiple filtering steps to ensure candidate specificity. The screening suggested two potential candidates that were finally assayed in the human microglial cell line HMC3. Upon stimulation with anti-TREM2 mAb, pSYK/SYK ratios were calculated in the presence of the candidates. As a result, we found that varenicline, a smoking cessation medication, can be considered as a transmembrane agonist of the TREM2-TYROBP interaction.

molecular biology↗

bioRxiv2024

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

developmental biology↗

bioRxiv2023

Row1, a member of a new family of conserved fungal proteins involved in infection, is required for appressoria functionality in Ustilago maydis

The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with non-melanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria-specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decrease appressoria formation and plant penetration, thereby reducing virulence. Specifically, the {Delta}row1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the {Delta}row1 mutant has secretion defects. Our data suggest that Row1 could modify the glucans that form the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family.

microbiology↗