Forests in the Appalachian Mountains are downwind from coal-fired power plants and other industrial sources in the Midwest, thus forests are exposed to relatively high concentrations of metal pollutants. Despite heightened public concern and tighter regulations to reduce anthropogenic pollutants, spruce-fir forests in the Appalachian Mountains are declining. In previous work at our study site, elevated phytochelatins were correlated with red spruce (Picea rubens) forest decline by altitude. Phytochelatins are metal-binding peptides synthesized from glutathione, from the precursor cysteine. Phytochelatins provide a sensitive and direct indication of metals stress at the cellular level. Metal analysis in soils can reflect historical metals deposition, while lichens can reflect recent deposition. In previous research, we studied metal stress and phytochelatin production in P. rubens from six mountains in the Appalachian Mountains. We found that patterns of foliar uptake and PC production are complex, but appear consistent with induction by Zn and Cd in organic soil. Our current purpose was to collect data along an elevational gradient on Whiteface Mountain, New York to supplement the east-west transect data collected previously and to characterize mycorrhizal fungus communities on sapling and mature tree roots. In recent work, we i) explored elevation patterns in the dominance of mycorrhizal species using PCR, DNA sequencing, and BLAST search; ii) measured Cd, Cu, Hg, Pb, and Zn in organic soil, mineral soil and metals uptake in P. rubens foliage and lichens; and iii) assessed P. rubens phytochelatin, glutathione, and cysteine production in order to monitor ongoing changes in red spruce response to metals deposition. While there was a consistent trend of increasing Pb and Zn with elevation in mineral soils, reflecting historic deposition patterns, there was no evidence for any systematic variation in metals content of foliage with elevation. Phytochelatin and mycorrhizal analyses are ongoing.

After removing the Elwha dam, sediments were exposed which pose a challenge for revegetation, due to lack of nutrients and organic matter. Ectomycorrhizal fungi (EMF) are known to facilitate uptake of nutrients and water and therefore effective colonization of seedlings would be expected to promote seedling establishment, survival and growth. Seedlings were planted in nursery potting soil mixed with local forest soil, compared to potting soil alone, to determine whether this practice will promote colonization and diversity of EMF on Abies grandis (grand fir) and Pseudotsuga menziesii (Douglas-fir) seedlings before outplanting at the Elwha restoration site. EMF species will be identified by examining morphology and then, for each morphotype, DNA extraction, PCR using ITS-1F and ITS-4 primers, and sequence analysis will be used to compare sequences to published fungal sequences using the BLAST algorithm. While molecular analyses are not yet complete, preliminary analysis of morphotypes revealed a consistent trend of increased diversity due to inoculation, based on an increased number of unique morphotypes per seedling, from 3.8 to 4.5 for grand fir, which was not significant, and from 4.2 to 5.6 for Douglas-fir, which was weakly significant (2 tailed t-test, p=0.093). This suggests that pre-inoculation with forest soil may promote colonization and therefore aid in restoration efforts.

Glucose and glutamine are primary carbon sources for cells to produce biomolecules and energy. Increased aerobic glycolysis, de novo fatty acid synthesis (FAS), and glutaminolysis are hallmarks of cancer cell metabolism and many tumors are dependent upon these for their survival. Likewise, virally infected cells also have considerably altered metabolic requirements. Our lab studies Kaposi’s Sarcoma Herpes Virus (KSHV), the etiologic agent of Kaposi’s Sarcoma (KS). KS is the most common tumor found in AIDS patients. Previous work from our lab showed that latent KSHV infected endothelial cells also require increased aerobic glycolysis and FAS. Our lab also published a global metabolic analysis of latent KSHV infection, revealing that latent KSHV infection also induces increased glutamine levels. Exogenous glutamine is metabolized through Glutaminolysis; glutamine is converted to glutamate and alpha-ketoglurarate (αKG) through two key enzymes, glutaminase (GLS) and glutamate dehydrogenase (GDH). Further, αKG can enter the tricarboxylic acid cycle (TCA) and support ATP production. Cancer cells utilize glutamine to replenish the TCA cycle when glucose is primarily used to produce lactate and fatty acids. Preliminary data shows that exogenous glutamine is required for latent KSHV infection through starving infected endothelial cells of glutamine. While glutamine starvation does not reduce viability of mock-infected endothelial cells, the absence of glutamine leads to significant cell death in KSHV infected cells. I hypothesize that glutamine is required during latent KSHV infection to replenish the TCA cycle. To study this, I inhibited glutaminolysis using a compound called BPTES, an inhibitor of GLS. Inhibiting glutaminolysis also leads to significant cell death of KSHV infected cells. This data indicates that glutamine is required during latent KSHV infection to replenish the TCA cycle. In the future I will add back TCA cycle intermediates to rescue cell death. Through our metabolomics studies of latent KSHV infection, we are identifying potential targets for KS therapy.