Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture

Starvation causes disturbance in amino acid and fatty acid metabolism in \u3ci\u3eDiporeia\u3c/i\u3e

The benthic amphipod Diporeia spp. was once the predominant macroinvertebrate in deep, offshore regions of the Laurentian Great Lakes. However, since the early 1990s, Diporeia populations have steadily declined across the area. It has been hypothesized that this decline is due to starvation from increasing competition for food with invasive dreissenid mussels. In order to gain a better understanding of the changes in Diporeia physiology during starvation, we applied two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCXGC/TOF-MS) for investigating the responses in Diporeia metabolome during starvation. We starved Diporeia for 60 days and collected five organisms every 12 days for metabolome analyses. Upon arrival to the laboratory, organisms were flash frozen and served as control (day 0). We observed an increase in lipid oxidation and protein catabolism with subsequent declines of essential amino acids (proline, glutamine, and phenylalanine), down-regulation of glycerophospholipid and sphingolipid metabolism, and decreased polyunsaturated fatty acid abundance in nutritionally stressed Diporeia. Abundance of 1-Iodo-2- methylundecane, a metabolite closely related to insect pheromones, also declined with starvation. This research has further substantiated the applicability of GCXGC/TOF-MS as a research tool in the field of environmental metabolomics. The next step is to apply this new knowledge for evaluating nutritional status of feral Diporeia to elucidate the underlying cause(s) responsible for their decline in the Great Lakes

Metabolite Profiles in Starved \u3ci\u3eDiporeia\u3c/i\u3e Spp. Using Liquid Chromatography-Mass Spectrometry (LC-MS) Based Metabolomics

The holarctic amphipod Diporeia spp. was historically the most abundant benthic macroinvertebrate in the offshore region of the Laurentian Great Lakes basin. However, since the 1990’s, the numbers of Diporeia have declined precipitously throughout the region. Competition for food with introduced dreissenid mussels may be partly to blame for this decline. Thus, a better understanding of how Diporeia responds and adjust to starvation is needed. For this purpose, we used liquid chromatography (LC) coupled with time-of-flight mass spectrometry (TOFMS) to study the metabolite profiles of Diporeia during starvation. Diporeia were collected from Lake Michigan, brought to the laboratory and starved for up to 60 days. During the starvation period, metabolite levels were determined at 12-day intervals and compared to those of day 0. Principal component and cluster analyses revealed differential abundance of metabolite profiles across groups. Significantly down-regulated metabolites included polyunsaturated fatty acids, phospholipids, and amino acids and their derivatives. Overall, starved organisms relied predominantly on glycerophospolipid metabolism and protein based catabolism for energy production. This research demonstrates that LC-MS based metabolomics can be used to assess physiological status and has shown that unique metabolite profiles are distinguishable over several weeks of starvation in this freshwater amphipod. More importantly, these unique metabolites could be used to gain insights into the underlying cause(s) of Diporeia’s decline in the Laurentian Great Lakes

Metabolite Profiles in Starved \u3ci\u3eDiporeia\u3c/i\u3e Spp. Using Liquid Chromatography-Mass Spectrometry (LC-MS) Based Metabolomics

The holarctic amphipod Diporeia spp. was historically the most abundant benthic macroinvertebrate in the offshore region of the Laurentian Great Lakes basin. However, since the 1990’s, the numbers of Diporeia have declined precipitously throughout the region. Competition for food with introduced dreissenid mussels may be partly to blame for this decline. Thus, a better understanding of how Diporeia responds and adjust to starvation is needed. For this purpose, we used liquid chromatography (LC) coupled with time-of-flight mass spectrometry (TOFMS) to study the metabolite profiles of Diporeia during starvation. Diporeia were collected from Lake Michigan, brought to the laboratory and starved for up to 60 days. During the starvation period, metabolite levels were determined at 12-day intervals and compared to those of day 0. Principal component and cluster analyses revealed differential abundance of metabolite profiles across groups. Significantly down-regulated metabolites included polyunsaturated fatty acids, phospholipids, and amino acids and their derivatives. Overall, starved organisms relied predominantly on glycerophospolipid metabolism and protein based catabolism for energy production. This research demonstrates that LC-MS based metabolomics can be used to assess physiological status and has shown that unique metabolite profiles are distinguishable over several weeks of starvation in this freshwater amphipod. More importantly, these unique metabolites could be used to gain insights into the underlying cause(s) of Diporeia’s decline in the Laurentian Great Lakes

Elucidating Causes of \u3ci\u3eDiporeia\u3c/i\u3e Decline in the Great Lakes via Metabolomics: Physiological Responses after Exposure to Different Stressors

The benthic macroinvertebrate Diporeia spp. have been extirpated from many areas of the Laurentian Great Lakes, but the mechanisms underlying such declines are not fully understood. Diporeia declines coinciding with the invasion of exotic dreissenid mussels (zebra and quagga) have led to the hypothesis that Diporeia declines are a result of decreased food availability from increasing competition with dreissenids for diatoms. There is additional evidence that Diporeia are negatively affected when in close proximity to dreissenids, probably because of exposure to toxins present in the mussels’ pseudofeces. Diporeia are also known to be sensitive to anthropogenic contaminants (such as polychlorinated biphenyls [PCBs]) present in Great Lakes sediments. To better understand the physiological responses of Diporeia to diverse stressors, we conducted three 28-d experiments evaluating changes in the metabolomes of Diporeia (1) fed diatoms (Cyclotella meneghiniana) versus starved, (2) exposed (from Lake Michigan and Cayuga Lake) to quagga mussels (Dreissena bugensis), and (3) exposed to sediments contaminated with PCBs. The metabolomes of samples were examined using both two-dimensional gas and liquid chromatography coupled with mass spectrometry. Each stressor elicited a unique metabolome response characterized by enhanced citric acid cycle, fatty acid biosynthesis, and protein metabolism in diatom-fed Diporeia; impaired glycolysis, protein catabolism, and folate metabolism in Diporeia from Lake Michigan irrespective of quagga mussel exposure, suggesting lakespecific adaptation mechanisms; and altered cysteine and phospholipid metabolism during PCB exposure. Subsequent comparisons of these stressor-specific metabolic responses with metabolomes of a feral Diporeia population would help identify stressors affecting Diporeia populations throughout the Great Lakes

Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes - Fig 3

<p><b>Principal component analysis reveals clustering of (A) Mexican serum samples by infecting DENV serotype and (B) Nicaraguan serum samples by primary or secondary infection.</b> In A, red spheres represent patients infected with DENV-1, and blue spheres represent patients infected with DENV-2. The percentage of variation found is: X axis 19.65%, Y axis 12.43%, Z axis 10.09%. In B, red spheres represent patients with primary-infection; blue spheres represent patients with secondary infection. The percentage of variation found is: X axis 31.75%, Y axis 10.72%, Z axis 8.83%</p

The Lipid repertoire of DENV infected mosquito cells is unfavorable for replication in the presence of C75.

<p>The specific lipid classes differentially regulated by C75 treatment of cells are shown. Panel A represents the fold changes of lipid classes expressed in DENV infected cells (MOI 3) compared to C75 treated DENV infected cells. Panels B–D show fold changes for individual molecular species within each lipid class that are regulated. PI, phosphatidylinositol; PG, phosphatidylglycerol; MG, monoacylglycerol; DG, diacylglycerol; PA, phosphatidic acid. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat.1002584.s006" target="_blank">table S3</a> for a complete list of lipid features detected in this analysis and supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002584#ppat-1002584-g002" target="_blank">figure 2</a> for a heat map representation of the data. Three replicates were included in the lipidomic analyses.</p

Newly synthesized lipids and viral RNA in subcellular fractions show a dynamic distribution with time of infection.

<p><b>A.</b> A pulse-chase analysis of ¹⁴C-acetate incorporation into newly synthesized lipids. The results show total labeled lipid in post-nuclear supernatants of C6/36 cells infected with DENV for 36 and 60 hr at an MOI of 20. <b>B.</b> The same pulse-chase analysis showing ¹⁴C-acetate incorporation into newly synthesized lipids in subcellular fractions (16K and CE) at 36 and 60 hr post-infection. <b>C.</b> The ratio of viral RNA genome copies per labeled lipid in subcellular fractions (described in B) at 36 and 60 hr post-infection. 16K, membrane fraction (pellet) following centrifugation of post-nuclear supernatants at 16, 000× g. CE, cytoplasmic extract following centrifugation of post-nuclear supernatants at 16,000× g. cpm, counts per minute.</p