Systems-Scale Analysis Reveals Pathways Involved in Cellular Response to Methamphetamine

Background: Methamphetamine (METH), an abused illicit drug, disrupts many cellular processes, including energy metabolism, spermatogenesis, and maintenance of oxidative status. However, many components of the molecular underpinnings of METH toxicity have yet to be established. Network analyses of integrated proteomic, transcriptomic and metabolomic data are particularly well suited for identifying cellular responses to toxins, such as METH, which might otherwise be obscured by the numerous and dynamic changes that are induced. Methodology/Results: We used network analyses of proteomic and transcriptomic data to evaluate pathways in Drosophila melanogaster that are affected by acute METH toxicity. METH exposure caused changes in the expression of genes involved with energy metabolism, suggesting a Warburg-like effect (aerobic glycolysis), which is normally associated with cancerous cells. Therefore, we tested the hypothesis that carbohydrate metabolism plays an important role in METH toxicity. In agreement with our hypothesis, we observed that increased dietary sugars partially alleviated the toxic effects of METH. Our systems analysis also showed that METH impacted genes and proteins known to be associated with muscular homeostasis/ contraction, maintenance of oxidative status, oxidative phosphorylation, spermatogenesis, iron and calcium homeostasis. Our results also provide numerous candidate genes for the METH-induced dysfunction of spermatogenesis, which have not been previously characterized at the molecular level. Conclusion: Our results support our overall hypothesis that METH causes a toxic syndrome that is characterized by the altered carbohydrate metabolism, dysregulation of calcium and iron homeostasis, increased oxidative stress, and disruption of mitochondrial functions

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

Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and -resistant \u3ci\u3eDrosophila melanogaster\u3c/i\u3e strains in response to DDT and oxidative stress

Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been associated with resistance to oxidative stress. However, little is known in regards to the impact of xenobiotics on induction of P450s and GSTs and if there exist differences in inducibility between the pesticide susceptible and resistant strains. Thus, we investigated the transcriptional expression of GSTs and P450s in DDT resistant (Wisconsin) and susceptible (Canton-S) Drosophila strains in response to exposure to DDT and the oxidative stressor H2O2. Wisconsin constitutively over-transcribed P450s, constitutively under-transcribed 27% of its total GSTs, and was more susceptible to H2O2 than Canton-S. DDT exposure induced GST expression only in the Wisconsin strain and not in the Canton-S strain. These results suggest that there are potentially more differences between pesticide susceptible and resistant strains than just constitutive expression of P450s; there may also exist, at least in some strains, differences in their patterns of inducibility of P450s and GSTs. Within the context of the Wisconsin strain, these differences may be contributing to the fly lines increased susceptibility to oxidative stress

Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D\u3csub\u3e2\u3c/sub\u3e, vitamin D\u3csub\u3e3\u3c/sub\u3e, 25- hydroxyvitamin D\u3csub\u3e2\u3c/sub\u3e and 25-hydroxyvitamin D\u3csub\u3e3\u3c/sub\u3e

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define “optimal” vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid–liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/ mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9–111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25 hydroxyvitamin D3 molecules in biological samples