High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah−/− mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease

Lipid Droplet Motility Increases Following Viral Immune Stimulation

Lipid droplets (LDs) have traditionally been thought of as solely lipid storage compartments for cells; however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle–organelle interactions. Their dynamics are known to change during cellular stress or nutrient deprivation; however, their movement during pathogen infections, especially at very early timepoints, is under-researched. This study aimed to track LD dynamics in vitro, in an astrocytic model of infection. Cells were either stimulated with a dsRNA viral mimic, poly I:C, or infected with the RNA virus, Zika virus. Individual LDs within infected cells were analysed to determine displacement and speed, and average LD characteristics for multiple individual cells calculated. Both LD displacement and mean speed were significantly enhanced in stimulated cells over a time course of infection with an increase seen as early as 2 h post-infection. With the emerging role for LDs during innate host responses, understanding their dynamics is critical to elucidate how these organelles influence the outcome of viral infection

LD content alters the regulation of ISGs in a cell-type dependent manner.

<p><b>A.</b> Huh-7 and HeLa cells were transiently transfected 24 hrs prior to stimulation with 500 ng/well of ISRE-Luc and 5 ng/well of pRL-TK in 12-well tissue culture plates. Luciferase measurements were taken 5 hrs post stimulation with IFN-β (ns = not statistically significant). <b>B.</b> HeLa and <b>C.</b> Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to stimulation with 1000 units/ml of. IFN-β for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFIT-1 (ii) OAS-1 and (iii) Viperin (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).</p

Lipid droplet content alters the host cell response to sendai virus.

<p> Hela cells were pre-incubated in either low serum or control media for 48 hrs prior to infection with 50 HAU/ ml SeV. Following 8 and 24 hrs infections of SeV, RTq-PCR was performed to quantitate mRNA expression of <b>A.</b> IFN-β and <b>B.</b> IFN-λ. (*, <i>p<</i> 0.05; **, <i>p<</i> 0.01; ***, <i>p<</i> 0.001).</p

Reduced lipid droplet content in cells reduces and delays the host response to dsDNA.

<p><b>A.</b> HeLa and <b>B.</b> Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to transfection with 0.5 μg poly dA:dT per well for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFN-β and (ii) IFN- λ or <b>C.</b> Viperin mRNA (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).</p

Lipid droplet mass is reduced by using lowered serum media, with little effect on their biogenesis.

<p><b>A.</b> Reduced serum (10% to 2% FCS) was applied to Huh-7 and HeLa cells for 48 hrs. Bodipy 505/515 (i) and Oil Red O (ii) were used to stain neutral lipid in both Huh-7 and HeLa cells. Images of fixed cells were captured using a Nikon Eclipse Ti-E microscope at 20X and 40X magnification respectively. DAPI counterstaining was also used to visualise cell nuclei. <b>B.</b> Following visualisation of bodipy stained cells, quantification of fluorescence intensity was performed using NIS Elements AR v.3.22. ***p < 0.001 <b>C.</b> Cells were grown in low serum conditions (2% FCS) for 72 hrs. Rapamycin (RAPA) and Chloroquine (CQ) were used as a positive control for the induction of autophagy. Membranes were probed with anti-LC3 specific antibody and anti-rabbit HRP. Membranes scanned using Amersham 600 chemiluminescence imager (i); Densitometry was performed using Image J analysis (ii) <b>D.</b> LD number was reduced in Huh-7 and HeLa cells by reducing FCS in media to 2% for 48 hrs prior to experiment, representative by time 0. Cells were then returned to 10% FCS media at commencement for experiment, and fixed at the indicated time points. Cells were stained with Bodipy 505/515 and DAPI prior to imaging using a Nikon Eclipse Ti-E microscope at 20X magnification (i), and subsequent image analysis using NIS elements (ii).</p

Lipid droplet content does not impact nucleic entry into Huh-7 cells.

<p><b>A. A.</b> Huh-7 cells were either incubated in control media or 2% FCS media, 48 hrs prior to transfection with rhodamine conjugated poly I:C. <b>B.</b> Cells were imaged, using a Nikon T<i>i-</i>E inverted microscope and quantification of fluorescence intensity performed using NIS Elements AR v.3.22.</p

Primer table.

<p>Primer table.</p

Cellular communication through extracellular vesicles and lipid droplets

Abstract Cellular communication is essential for effective coordination of biological processes. One major form of intercellular communication occurs via the release of extracellular vesicles (EVs). These vesicles mediate intercellular communication through the transfer of their cargo and are actively explored for their role in various diseases and their potential therapeutic and diagnostic applications. Conversely, lipid droplets (LDs) are vesicles that transfer cargo within cells. Lipid droplets play roles in various diseases and evidence for their ability to transfer cargo between cells is emerging. To date, there has been little interdisciplinary research looking at the similarities and interactions between these two classes of small lipid vesicles. This review will compare the commonalities and differences between EVs and LDs including their biogenesis and secretion, isolation and characterisation methodologies, composition, and general heterogeneity and discuss challenges and opportunities in both fields

Illuminating the liminality of the doctoral journey:precarity, agency and COVID-19

There is an academic acceptance that doctoral studies are a complex, multifaceted endeavour bound with the differing, emerging and contrasting identities of the students who undertake them. This article explores such journeys in the context of one of the most disruptive events to hit the higher educational sector worldwide: COVID-19. The study utilises data collected via an online forum established to collectively explore the challenges, the opportunities and the tensions in the lives of 12 students enrolled in a doctoral degree in an Australian higher education institution. Results indicate that the journey of such students pre-COVID-19 was characterised by a sense of liminality, agency and journey. COVID-19 exacerbated such feelings. The conclusion points to a need to enhance spaces of agency in the graduate research experience to reflect the multidimensionality of students’ lives.</p