Olympic mudminnow (Novumbra hubbsi), Washington’s only endemic fish species, was listed as “Sensitive” at the state level in 1999. However, limited knowledge and understanding has stalled conservation efforts. This study investigates the impact of invasive fishes on Olympic mudminnnow, focusing on Lake Ozette in Olympic National Park. Using stable isotopes from the species present in Lake Ozette, the trophic ecology of mudminnow was determined. Stable isotopes are naturally occurring and can be used to trace the pathways of aquatic- and terrestrial-derived energy and carbon transfer in aquatic ecosystems. This analysis method investigates carbon (¹³C, ¹²C) and nitrogen (¹⁵N, ¹⁴N) found within samples. The presence of these isotopes depends on what a species eats. Moving up the food chain, the ratios of ¹³C/¹²C and ¹⁵N/¹⁴N increase, identifying a species’ feeding position within a community. The higher the trophic position, the more ¹³C and more ¹⁵N will be present in the sample. Through analysis of the entire community, including fish, invertebrates, and plants, a generalized food web was constructed for Lake Ozette. Using linear models and a permutation method, the fish species were compared to the model to test for statistically significant differences between the measured ratio of carbon to nitrogen. If the position of the fish species was significantly different from the model prediction, then there is no niche overlap and competition is unlikely. The combined results of the stable isotope and spatial overlap analyses indicated which species impact Olympic mudminnow within Lake Ozette. This study provided key information concerning complex predator-prey interactions, which must be understood to manage Olympic mudminnow as well as the invasive species present in Lake Ozette. Since there is little known concerning this species, there are many opportunities to extend the knowledge base continuing both with the predator prey interactions and with other knowledge gaps.

Transporters are membrane proteins that modulate the absorption, disposition, and clearance of xenobiotics, and they are potentially involved in adverse drug reactions (ADRs). In the liver, which is the major organ for xenobiotic biotransformation, uptake transporters bring substrates into hepatocytes for further processing, whereas efflux transporters eliminate substrates out of the cell. In predicting pharmacokinetic effects, regulation of transporters may determine the availability of substrates to intracellular enzymes and affect the toxicological responses to xenobiotics. During development, profound changes occur in the expression of transporters, and understanding the age effect is crucial in predicting the efficacy and toxicity of therapeutic drugs for the pediatric population. Furthermore, age profoundly alters the gut microbiome, and this may alter certain microbial metabolites and modify the host response. Very little is known regarding the relationship between transporter regulation and gut microbiome during development. Therefore, the goal of this study was to determine the developmental regulation of transporters using germ-free (GF) mice as a model system. Both conventional (CV) and GF wild-type mice were bred under the same housing conditions, and livers were collected at the following seven ages: Day 1, 5, 10, 15, 25, 60, and 120 (n=5 per age per sex). RNA was isolated from the liver and the mRNA expression was determined using RT-qPCR. Significance was determined using a T-Test between age-matched CV and GF by sex. Our data show that expression levels of Abcg5 and Abcg8 are consistently higher in male GF than CV mice. For Abcg5, GF males at days 1, 60 and 120 and for Abcg8, GF males at days 1, 10, and 120 had significantly higher expression levels than CV mice. Our result suggests that liver transporters may in part be regulated in a microbiome- dependent manner.

Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 200 nucleotides. Recent studies have recognized lncRNAs as crucial regulators of transcription, mRNA processing and protein activity. However, little is known regarding the pharmacological regulation of lncRNAs in liver, which is an important organ for xenobiotic biotransformation. The goal of this study was to investigate the correlation between lncRNAs and the constitutive androstane receptor (CAR/Nr1i3), which is a liver-enriched xenobiotic-sensing nuclear receptor that plays an important role in xenobiotic biotransformation and intermediary metabolism. Three genotypes of mice, namely wild-type (WT), CAR-null and humanized CAR-transgenic (hCAR-TG) mice at 5- or 60-days of age were administered a species-appropriate CAR ligand (TCPOBOP for mCAR, and CITCO for hCAR) or vehicle once daily for 4-days, and livers were removed on the 5th day. The lncRNA gene expression was determined using RNA-Seq (n=3 per group, Cuffdiff, FDR-BH<0.05). At Day 5 neonatal age, mCAR activation altered more lncRNAs than hCAR activation (877 vs. 234). Interestingly, only 87 lncRNAs were co-regulated by both mCAR and hCAR, whereas 790 were only regulated by mCAR, and 147 were only regulated by hCAR. At Day 60, mCAR activation also altered more lncRNAs than hCAR activation. Among the differentially regulated lncRNAs, 126 lncRNAs were co-regulated, 1110 and 87 were uniquely altered by mCAR and hCAR respectively. The results also showed the significance of age in the expression levels of lnRNAs. For example, mCAR activation produced more changes in lncRNA expression at Day 60 than at Day 5; however, his pattern was not observed in hCAR-TG mice. This study highlights the age and the species differences of the lncRNA regulation in liver following pharmacological activation of CAR. The differentially regulated lncRNAs may serve as novel biomarkers or therapeutic targets for human diseases such as metabolic syndrome and drug-induced liver injuries.

Newborns and children are at a much higher risk to adverse drug reactions, which is a significant public health problem. This is partially due to the lack of knowledge of the ontogeny of drug-metabolizing enzymes (DMEs). There are two types of DMEs, known as Phase-I and Phase -II DMEs. Phase I DMEs catalyze oxidation, reduction and hydrolysis reactions. Cytochrome P450s (CYPs) are a superfamily of Phase I oxidative enzymes responsible for the metabolism of a wide spectrum of drugs. Phase-II DMEs are responsible for various conjugation reactions including UDP glucuronidation, sulfonation and glutathione conjugation. Gut microbiome is known to regulate xenobiotic metabolism in adult mouse liver, but little is known of its potential involvement in the ontogeny of DMEs. The goal of this study was to determine the developmental regulation of Phase I and Phase II DMEs using germ free (GF) mice as a model. We bred both control and GF mice under the same housing conditions, and collected livers from male mice at the following ages: Days 1, 5, 10, 15, 25, 60, and 120. We isolated total RNAs and determined the RNA concentration using a Nanodrop spectrophotometer. We also determined the RNA integrity using gel electrophoresis by visualizing the 28S and 18S ribosomal RNA subunits. Using RT-qPCR we quantified the mRNAs of major DMEs and evaluated differences between control and GF mice using T-Test. As compared to age-matched control mice, the mRNAs of the xenobiotic-metabolizing Cyp3a11 was down-regulated in livers of GF mice at multiple ages including Days 10, 60, 120. Conversely, the expression of the lipid-metabolizing enzyme Cyp4a14 was up-regulated at the following ages: Days 5, 15, 60, and 120 in GF mice. In conclusion, our data suggest that the ontogeny of some DMEs is profoundly modified by lack of gut microbiota.

The hippocampus, a brain area traditionally thought to play a key role in memory consolidation, has been shown to be involved in encoding contextual information during reward-motivated decision-making tasks. Projections to and from the hippocampus connect it with structures known to be involved in decision-making behavior and encoding cost/benefit values, making it well situated to contribute to the circuitry processing choice selection. Inactivation of hippocampal areas in rats have been shown to increase variability of choice preference between large rewards with an associated delay, and small rewards received immediately. To elucidate the role of the hippocampus in encoding economic factors of rewards, we recorded from place cells in a hippocampal subregion known to be necessary for spatial coding of salient objects and events, called CA1, during a delay-discounting maze-based task. The task was designed on an elevated T-maze to have three blocks during each daily recording session. During each block the rat chooses several times between a large reward (4 sugar pellets) with a long delay specific to that block (10, 20, or 40 seconds) and a small reward (1 sugar pellet) with a short delay of 3 seconds across all blocks. The rats were familiarized with the delays at the beginning of each block by undergoing 5 forced trials on each side. Behavior measured over 10 free choice trials then completed the block. As expected, preference for the large reward was maximal during 10-second blocks and declined as the delay increased. Preliminary analysis of place fields shows remapping between different delay conditions indicating spatial encoding of reward costs by hippocampus. Remapping was also observed between free and forced trials. These results imply that the place field representation of maze areas reflects contextual spatial information that includes the expected cost of rewards.