Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Background: Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results: Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion: Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.Plymouth State University Graduate Programs OfficeWoods Hole Oceanographic Institution Academic Programs OfficeNew Hampshire IDeA Network of Biological Research Excellence (NH-INBRE)National Center for Research Resources (U.S.) (Grant 5P20RR030360-03)National Institute of General Medical Sciences (U.S.) (Grant 8P20GM103506-03

Bioinformatic and Gene Expression Analysis of an SLC6 Homolog in the Toxin-producing Marine Diatom Pseudo-nitzschia Multiseries

Pseudo-nitzschia multiseries (Ps-n) is a marine pennate diatom that produces the neurotoxin, domoic acid (DA). The molecular processes involved in toxin synthesis have yet to be elucidated. Previous work generated a suite of candidate genes that were found to be up-regulated under toxin-producing conditions. One such gene showed high sequence homology to the SLC6 family of amino acid transporters, which have been found to transport an array of amino acid substrates. This thesis focused on further characterizing the gene structure and expression patterns of this gene which we have designated PSN0072. Sequence analysis of the predicted protein revealed a conserved domain found in the SLC6 transporter family. Alignment of this amino acid sequence with a functional alignment of SLC6 homologs revealed the conservation of functionally relevant residues, strengthening the putative identification of PSN0072 as a member of the SLC6 family. Gene expression analyses revealed a link between the regulation of this gene and DA levels. Interestingly, PSN0072 was also up-regulated under nitrogen limited conditions, a finding consistent with other characterized amino acid importers. The results presented in this thesis suggest that PSN0072 may be involved in the export of DA or the import of another amino acid substrate, either as a nitrogen source or as a precursor to DA synthesis. A possible hypothesis to explain the results presented is that PSN0072 may be able to perform both of these important functions to Ps-n physiology

Assessing the Perceptions of Fibromyalgia Syndrome in United States Among Academic Physicians and Medical Students: Where are We and Where are We Headed?

Objectives: Fibromyalgia syndrome [FMS] is a common condition, often diagnosed in the primary care setting, causing diffuse pain with additional somatic symptoms. Many physicians have questioned the existence of FMS due to an unclear pathophysiological origin and its overlap with other somatic syndromes. We sought to assess the perceptions of FMS among United States medical students and internal medicine and family medicine trained physicians working in an academic hospital. Methods: Residents and attendings working in a local teaching hospital were given questionnaires during undergraduate medical education sessions and academic conferences in internal medicine and family medicine. Medical students received surveys during small group sessions and through student mailboxes. Results: Seventy-two internal and family medicine trained physicians and 211 medical students were surveyed. In assessing whether FMS was primarily physiological or psychological in origin, 66% of physicians compared to only 29% of medical students [p < 0.001] chose a psychological etiology. Among physicians, women [82%] and American medical graduates [77%] were more likely to endorse a psychological mechanism than their counterparts [53% p < 0.010, 50% p = 0.022, respectively]. Additionally, when physicians were asked whether they believed FMS was an "actual illness," 19% responded "no." Conclusions: Fibromyalgia syndrome continues to be a controversial illness in the United States, especially when compared with values attained from international studies. Although current academic physicians appear to be skeptical of FMS's existence or its potential physiological mechanisms, medical students are more likely to support a physiological mechanism