Opposing action of the FLR-2 glycoprotein hormone and DRL-1/FLR-4 MAP kinases balance p38-mediated growth and lipid homeostasis in C. elegans.

Animals integrate developmental and nutritional signals before committing crucial resources to growth and reproduction; however, the pathways that perceive and respond to these inputs remain poorly understood. Here, we demonstrate that DRL-1 and FLR-4, which share similarity with mammalian mitogen-activated protein kinases, maintain lipid homeostasis in the C. elegans intestine. DRL-1 and FLR-4 function in a protein complex at the plasma membrane to promote development, as mutations in drl-1 or flr-4 confer slow growth, small body size, and impaired lipid homeostasis. To identify factors that oppose DRL-1/FLR-4, we performed a forward genetic screen for suppressors of the drl-1 mutant phenotypes and identified mutations in flr-2 and fshr-1, which encode the orthologues of follicle stimulating hormone and its putative G protein-coupled receptor, respectively. In the absence of DRL-1/FLR-4, neuronal FLR-2 acts through intestinal FSHR-1 and protein kinase A signaling to restrict growth. Furthermore, we show that opposing signaling through DRL-1 and FLR-2 coordinates TIR-1 oligomerization, which modulates downstream p38/PMK-1 activity, lipid homeostasis, and development. Finally, we identify a surprising noncanonical role for the developmental transcription factor PHA-4/FOXA in the intestine where it restricts growth in response to impaired DRL-1 signaling. Our work uncovers a complex multi-tissue signaling network that converges on p38 signaling to maintain homeostasis during development.

Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.