Search

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) nucleases has transformed biotechnology by providing an easy, efficient, and versatile platform for editing DNA. However, traditional CRISPR-based technologies initiate editing by activating DNA double-strand break (DSB) repair pathways, which can cause adverse effects in cells and restrict certain therapeutic applications of the technology. To this end, several new CRISPR-based modalities have been developed that are capable of catalyzing editing without the requirement for a DSB. Here, we review three of these technologies: base editors, prime editors, and RNA-targeting CRISPR-associated protein (Cas)13 effectors. We discuss their strengths compared to traditional gene-modifying systems, we highlight their emerging therapeutic applications, and we examine challenges facing their safe and effective clinical implementation.

Microglia, the parenchymal immune cells of the central nervous system, orchestrate neuroinflammation in response to infection or damage, and promote tissue repair. However, aberrant microglial responses are integral to neurodegenerative diseases and critically contribute to disease progression. Thus, it is important to elucidate how microglia - mediated neuroinflammation is regulated by endogenous factors. Here, we explored the effect of Nerve Growth Factor (NGF), an abundant neurotrophin, on microglial inflammatory responses. NGF, via its high affinity receptor TrkA, downregulated LPS - induced production of pro-inflammatory cytokines and NO in primary mouse microglia and inhibited TLR4 - mediated activation of the NF-κB and JNK pathways. Furthermore, NGF attenuated the LPS - enhanced glycolytic activity in microglia, as suggested by reduced glucose uptake and decreased expression of the glycolytic enzymes Pfkβ3 and Ldhα. Consistently, 2DG - mediated glycolysis inhibition strongly downregulated LPS - induced cytokine production in microglial cells. Our findings demonstrate that NGF attenuates pro-inflammatory responses in microglia and may thereby contribute to regulation of microglia - mediated neuroinflammation.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.