Induction of a proliferative response in the zebrafish retina by injection of extracellular vesicles.

Ongoing research using cell transplantation and viral-mediated gene therapy has been making progress to restore vision by retinal repair, but targeted delivery and complete cellular integration remain challenging. An alternative approach is to induce endogenous Müller glia (MG) to regenerate lost neurons and photoreceptors, as occurs spontaneously in teleost fish and amphibians. Extracellular vesicles (EVs) can transfer protein and RNA cargo between cells serving as a novel means of cell-cell communication. We conducted an in vivo screen in zebrafish to identify sources of EVs that could induce MG to dedifferentiate and generate proliferating progenitor cells after intravitreal injection into otherwise undamaged zebrafish eyes. Small EVs (sEVs) from C6 glioma cells were the most consistent at inducing MG-derived proliferating cells. Ascl1a expression increased after intravitreal injection of C6 sEVs and knockdown of ascl1a inhibited the induction of proliferation. Proteomic and RNAseq analyses of EV cargo content were performed to begin to identify key factors that might target EVs to MG and initiate retina regeneration.

Repurposing N-acetylcysteine for management of non-acetaminophen induced acute liver failure: an evidence scan from a global health perspective.

Background: The World Health Organization (WHO)'s Essential Medicines List (EML) plays an important role in advocating for access to key treatments for conditions affecting people in all geographic settings. We applied our established drug repurposing methods to one EML agent, N-acetylcysteine (NAC), to identify additional uses of relevance to the global health community beyond its existing EML indication (acetaminophen toxicity). Methods: We undertook a phenome-wide association study (PheWAS) of a variant in the glutathione synthetase (GSS) gene in approximately 35,000 patients to explore novel indications for use of NAC, which targets glutathione. We then evaluated the evidence regarding biologic plausibility, efficacy, and safety of NAC use in the new phenotype candidates. Results: PheWAS of GSS variant R418Q revealed increased risk of several phenotypes related to non-acetaminophen induced acute liver failure (ALF), indicating that NAC may represent a therapeutic option for treating this condition. Evidence review identified practice guidelines, systematic reviews, clinical trials, retrospective cohorts and case series, and case reports. This evidence suggesting benefit of NAC use in this subset of ALF patients. The safety profile of NAC in this literature was also concordant with existing evidence on safety of this agent in acetaminophen-induced ALF. Conclusions: This body of literature indicates efficacy and safety of NAC in non-acetaminophen induced ALF. Given the presence of NAC on the EML, this medication is likely to be available across a range of resource settings; promulgating its use in this novel subset of ALF can provide healthcare professionals and patients with a valuable and safe complement to supportive care for this disease.

Depletion of METTL3 alters cellular and extracellular levels of miRNAs containing m6A consensus sequences.

Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs.

Rab13 regulates sEV secretion in mutant KRAS colorectal cancer cells.

Small extracellular vesicles (sEVs), 50-150 nm in diameter, have been proposed to mediate cell-cell communication with important implications in tumor microenvironment interactions, tumor growth, and metastasis. We previously showed that mutant KRAS colorectal cancer (CRC) cells release sEVs containing Rab13 protein and mRNA. Previous work had shown that disruption of intracellular Rab13 trafficking inhibits epithelial cell proliferation and invasiveness. Here, we show that Rab13 additionally regulates the secretion of sEVs corresponding to both traditional exosomes and a novel subset of vesicles containing both ß1-integrin and Rab13. We find that exposure of recipient cells to sEVs from KRAS mutant donor cells increases proliferation and tumorigenesis and that knockdown of Rab13 blocks these effects. Thus, Rab13 serves as both a cargo protein and as a regulator of sEV secretion. Our data support a model whereby Rab13 can mediate its effects on cell proliferation and invasiveness via autocrine and paracrine signaling.