Biology subjects

Lopez, D. M.

Publications and source records attributed to Lopez, D. M..

3 recordsLinked to original sources

bioRxiv2024

Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-inducedlung injury

Bronchopulmonary dysplasia (BPD), a chronic lung disease, is the most common major complication of preterm birth. Supplemental oxygen administration, while lifesaving in the neonatal period, remains a key determinant of BPD pathophysiology. Exposure of the immature lung to increased levels of oxygen elicits an inflammatory response resulting in abnormal lung development. However, not every premature infant is equally sensitive to develop BPD. Using genetically diverse mouse strains, we show that the innate immune response activated in the lungs of mice sensitive to hyperoxia that develop BPD-like lung injury differs from mice resilient to disease. Specifically, we identified a selective upregulation of triggering receptor expressed on myeloid cells 2 (TREM2) on lung macrophages and monocytes in the hyperoxia-sensitive C57BL/6J mouse strain. We show that loss of function of TREM2 signaling in myeloid cells resulted in a dramatically improved phenotype after neonatal hyperoxia exposure characterized by a dampened immune response, preserved alveolar structure, and preserved cell proliferative potential supporting normal lung development. At the molecular level, inhibition of TREM2 signaling dampened the magnitude of p53 activation and resulted in cell cycle arrest instead of apoptosis. These findings show that TREM2 is a critical regulator of the pathogenic innate immune response to hyperoxia and highlight its importance as a potential therapeutic target for mitigating injury in the hyperoxia-exposed developing lung.

immunology↗

bioRxiv2024

In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide-and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Forster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N-and C-terminal C atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices. STATEMENT OF SIGNIFICANCEBiomolecular structures involved in various biological processes, including muscle function and sensory perception, generate, convey, and respond to mechanical forces. In-vivo accurate measurement of these forces is challenging but needed to understand biological function. Here we present a comprehensive computational analysis of three different types of molecular force sensors used to report pico-Newton level forces in biomolecular systems. Our atom-level simulation predictions provide mechanistic insight that can facilitate experimental data interpretation, selection of sensor design parameters, and the development of new force sensors tailored to specific applications and environments.

biophysics↗

bioRxiv2021

CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000s of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei. Teaser SentenceCRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

bioengineering↗