A genome-wide CRISPR screen implicates plasma membrane asymmetry in exogenous C6-ceramide toxicity.

The bioactive sphingolipid ceramide impacts diverse cellular processes (e.g. apoptosis and cell proliferation) through its effects on membrane dynamics and intracellular signaling pathways. The dysregulation of ceramide metabolism has been implicated in cancer evasion of apoptosis and targeting ceramide metabolism has potential therapeutic benefits as a strategy to kill cancer cells and slow tumor growth. However, the mechanisms of cancer cell resistance to ceramide-mediated cell death are vastly intertwined and incompletely understood. To shed light on this mystery, we performed a genome-wide CRISPR-Cas9 screen to systematically identify regulators of cancer resistance to the soluble short chain ceramide, C6 ceramide (C6-Cer). Our results reveal a complex landscape of genetic modifiers of C6-Cer toxicity, including genes associated with ceramide and sphingolipid metabolism, vesicular trafficking, and membrane biology. Furthermore, we find that loss of the phospholipid flippase subunit TMEM30A impairs the plasma membrane trafficking of its binding partner, the P4-type ATPase ATP11B, and depletion of TMEM30A or ATP11B disrupts plasma membrane asymmetry and promotes resistance to C6-Cer toxicity. Together, our findings provide a resource of genetic modifiers of C6-Cer toxicity and reveal an unexpected role of plasma membrane asymmetry in C6-Cer induced cell death.

Increased interleukin-6 and macrophage chemoattractant protein-1 are associated with respiratory failure in COVID-19.

In SARS-CoV-2 infection there is an urgent need to identify patients that will progress to severe COVID-19 and may benefit from targeted treatment. In this study we analyzed plasma cytokines in COVID-19 patients and investigated their association with respiratory failure (RF) and treatment in Intensive Care Unit (ICU). Hospitalized patients (n = 34) with confirmed COVID-19 were recruited into a prospective cohort study. Clinical data and blood samples were collected at inclusion and after 2-5 and 7-10 days. RF was defined as PaO2/FiO2 ratio (P/F) < 40 kPa. Plasma cytokines were analyzed by a Human Cytokine 27-plex assay. COVID-19 patients with RF and/or treated in ICU showed overall increased systemic cytokine levels. Plasma IL-6, IL-8, G-CSF, MCP-1, MIP-1α levels were negatively correlated with P/F, whereas combinations of IL-6, IP-10, IL-1ra and MCP-1 showed the best association with RF in ROC analysis (AUC 0.79-0.80, p < 0.05). During hospitalization the decline was most significant for IP-10 (p < 0.001). Elevated levels of pro-inflammatory cytokines were present in patients with severe COVID-19. IL-6 and MCP-1 were inversely correlated with P/F with the largest AUC in ROC analyses and should be further explored as biomarkers to identify patients at risk for severe RF and as targets for improved treatment strategies.

¹9F magnetic resonance probes for live-cell detection of peroxynitrite using an oxidative decarbonylation reaction.

We report a newly discovered oxidative decarbonylation reaction of isatins that is selectively mediated by peroxynitrite (ONOO(-)) to provide anthranilic acid derivatives. We have harnessed this rapid and selective transformation to develop two reaction-based probes, 5-fluoroisatin and 6-fluoroisatin, for the low-background readout of ONOO(-) using (19)F magnetic resonance spectroscopy. 5-fluoroisatin was used to non-invasively detect ONOO(-) formation in living lung epithelial cells stimulated with interferon-γ (IFN-γ).

Dominant-negative Dmp53 extends life span through the dTOR pathway in D. melanogaster.

Expression of dominant-negative (DN) versions of the Drosophila ortholog of the tumor suppressor p53 extends fly life span in a Calorie Restriction (CR) dependent manner. DN-Dmp53 expression furthermore leads to reduction of Drosophila insulin-like peptide (dILP) 2 mRNA levels and a decrease in insulin/insulin-like growth factor-signaling activity (IIS) in the fly fat body. It is unclear by which mechanisms DN-Dmp53 extends longevity, and whether modulation of insulin-signaling activity plays a pivotal role in life span regulation by Dmp53. Here we show that life span extension due to DN-Dmp53 expression is likely due to reduction of Dmp53 activity and that decreased Dmp53 activity does not extend life span when dILP2 is concomitantly over expressed. Furthermore, extended longevity due to DN-Dmp53 expression does not further extend the life span of flies over expressing the IIS associated transcription factor dFoxO, indicating that DN-Dmp53-dependent life span extension may be related to IIS. However, reduction of dFoxO levels does not decrease DN-Dmp53-dependent longevity extension. Interestingly, when DN-Dmp53 is expressed in flies lacking the translation initiation controlling factor Thor/4E-BP, the downstream target of dTOR signaling, no increase in life span is observed. Taken together, these data suggest that Dmp53 may affect life span by differentially engaging the IIS and dTor pathways.

dSir2 and Dmp53 interact to mediate aspects of CR-dependent lifespan extension in D. melanogaster.

Calorie Restriction (CR) is a well established method of extending life span in a variety of organisms. In the fruit fly D. melanogaster, CR is mediated at least in part by activation of dSir2. In mammalian systems, one of the critical targets of Sir2 is the tumor suppressor p53. This deacetylation of p53 by Sir2 leads to inhibition of p53's transcriptional activity. We have recently shown that inhibition of Dmp53 activity in the fly brain through the use of dominant-negative (DN) constructs that inhibit DNA-binding can extend life span. This life span extension appears to be related to CR, as CR and DN-Dmp53 donot display additive effects on life span. Here we report that life span extension by DN-Dmp53 expression is highly dynamic and can be achieved even when DN-Dmp53 is expressed later in life. In addition, we demonstrate that life span extension by activation of dSir2 and DN-Dmp53 expression are not additive. Furthermore, we show that dSir2 physically interacts with Dmp53 and can deacetylate Dmp53-derived peptides. Taken together, our data demonstrate that Dmp53 is a down stream target of dSir2 enzymatic activity and mediates some aspects of the life span extending effects of CR.

Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling.

In Drosophila melanogaster, p53 (Dmp53) is an important mediator of longevity. Expression of dominant-negative (DN) forms of Dmp53 in adult neurons, but not in muscle or fat body cells, extends lifespan. The lifespan of calorie-restricted flies is not further extended by simultaneously expressing DN-Dmp53 in the nervous system, indicating that a decrease in Dmp53 activity may be a part of the CR lifespan-extending pathway in flies. In this report, we show that selective expression of DN-Dmp53 in only the 14 insulin-producing cells (IPCs) in the brain extends lifespan to the same extent as expression in all neurons and this lifespan extension is not additive with CR. DN-Dmp53-dependent lifespan extension is accompanied by reduction of Drosophila insulin-like peptide 2 (dILP2) mRNA levels and reduced insulin signaling (IIS) in the fat body, which suggests that Dmp53 may affect lifespan by modulating insulin signaling in the fly.