cGAS Mediates the Inflammatory Responses of Human Microglial Cells to Genotoxic DNA Damage.

Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.

Dynamic Nanostructures for Conditional Activation and Deactivation of Biological Pathways.

Nucleic acid nanotechnology utilizes natural and synthetic structural motifs to build versatile nucleic acid nanoparticles (NANPs). These rationally designed assemblies can be further equipped with functional nucleic acids and other molecules such as peptides, fluorescent dyes, etc. In addition to nucleic acids that directly interact with the regulated target gene transcripts, NANPs can display decoys, wherein the oligonucleotide stretches with transcription factor binding sequences, preventing transcription initiation. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a group of five crucial transcription factors regulating the pathogenesis of inflammatory diseases and cancer; as such, they are relevant targets for therapy. One therapeutic approach involves interdependent self-recognizing hybridized DNA/RNA fibers designed to bind NF-κB and prevent its interaction with the promotor region of NF-κB-dependent genes involved in inflammatory responses. Decoying NF-κB results in the inability to initiate transcription of regulated genes, showing a promising approach to gene regulation and gene therapy. The protocol described herein provides detailed steps for the synthesis of NF-κB decoy fibers, as well as their characterization using polyacrylamide gel electrophoresis (to confirm desired physicochemical properties and purity) and functional bioassays (to confirm desired biological activity).

An in-vitro quantitative investigation on the synergistic effect of capsaicin and 5-fluorouracil encapsulated into lipid nanocapsules to treat breast cancer.

BACKGROUND: Breast cancer conventional therapeutics are effective; however, they encounter some limitations including multidrug resistance, the presence of pharmacological barriers, and non-selectivity which hinder their optimal therapeutic efficacy. AIM: Overcoming such drawbacks necessitates the development of efficient drug vehicles including lipid-based nanoparticles. This study aimed to quantitatively investigate in-vitro the synergistic therapeutic effect of the novel combination of capsaicin and 5-fluorouracil (5-FU) encapsulated in lipid nanocapsules (LNCs). METHOD: To this end, thorough physicochemical and in-vitro assessments on the breast cancer cell line (MCF-7) were done. The drug-loaded LNCs were characterized using DLS, TEM imaging, stability study, and in-vitro release study. Furthermore, the biological activity of the prepared LNCs was assessed by implementing comparative cytotoxicity studies as well as apoptosis, and cell cycle flow cytometric analyses. RESULTS: The developed nanoformulations were monodisperse with average particle size (PS) of 31, 43.8, and 127.3 nm for empty LNCs, Cap-LNCs, and 5-FU-LNCs, respectively, and with a surface charge of -35.4, -21.7 and -31.4 mV, respectively, reflecting good physical stability. The TEM micrographs revealed the spherical morphology of the drugs-loaded LNCs with comparable PS to that obtained by DLS. on the other hand, all the biological assessments confirmed the superior antiproliferative effect of the combined drug-loaded LNCs over their free drug counterparts. CONCLUSION: Intriguingly, the study findings highlighted the potential synergistic activity of the drugs (capsaicin and 5-FU) and the extensive enhancement of their biological activity through incorporation into LNCs. Such promising results will pave the way to further novel combined nanoformulation in preclinical and clinical studies on breast cancer patients.