Report on Validation and Calibration of Fatty Acid Signatures in Blubber as Indicators of Prey in Hawaiian Monk Seal Diet

Pacific Islands Fisheries Science Center Administrative Reports are issued to promptly disseminate scientific and technical information to marine resource managers, scientists, and the general public. Their contents cover a range of topics, including biological and economic research, stock assessment, trends in fisheries, and other subjects. Administrative Reports typically have not been reviewed outside the Center. As such, they are considered informal publications. The material presented in Administrative Reports may later be published in the formal scientific literature after more rigorous verification, editing, and peer review. Other publications are free to cite Administrative Reports as they wish provided the informal nature of the contents is clearly indicated and proper credit is given to the author(s). Administrative Reports may be cited as follows: Iverson, S. J., B. S. Stewart, and P. K. Yochem. 2010. Report on validation and calibration of fatty acid signatures in blubber as indicators o

Carboxy-Terminal Truncation Activates glp-1 Protein to Specify Vulval Fates in Caenorhabditis elegans

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate

Utilization of the Clinical Laboratory for the Implementation of Concussion Biomarkers in Collegiate Football and the Necessity of Personalized and Predictive Athlete Specific Reference Intervals

Background: A continued interest in concussion biomarkers makes the eventual implementation of identified biomarkers into routine concussion assessment an eventual reality. We sought to develop and test an interdisciplinary approach that could be used to integrate blood-based biomarkers into the established concussion management program for a collegiate football team. Methods: We used a CLIA-certified laboratory for all testing and chose biomarkers where clinically validated testing was available as would be required for results used in clinical decision making. We summarized the existing methods and results for concussion assessment across an entire season to identify and demonstrate the challenges with the eventual integration of a parallel process using blood-based tests for concussion management. We analyzed the results of the biomarkers chosen for trends consistent with the outcome assessments provided from the current concussion management protocols. Results: Baseline samples were collected with three additional post-concussion samples collected at three separate time points from players with a diagnosed concussion (n = 12). A summary of results from currently used concussion assessment tools were compared to the representative biomarkers S100B and NSE results. Nine sport-related concussions occurred during practice and three during play. For S100B, 50 % had follow-up testing results lower than the post-injury result. In contrast, 92 % of NSE follow-up results were lower than post-injury. One hundred percent of the results for S100B and NSE were within the athlete-derived reference intervals upon return-to-play and season end. Conclusions: The reported workflow provides a framework for the eventual implementation of biomarkers for concussion assessment into existing assessment protocols and strengthens the need for reliance on clinical laboratory testing. Athlete-specific reference intervals will be required to adequately interpret results

Visualization of C. elegans transgenic arrays by GFP

BACKGROUND: Targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific DNA sequence, the lac operator (lacO), allows visualization of chromosomes in yeast and mammalian cells. In principle this method of visualization could be used for genetic mosaic analysis, which requires cell-autonomous markers that can be scored easily and at single cell resolution. The C. elegans lin-3 gene encodes an epidermal growth factor family (EGF) growth factor. lin-3 is expressed in the gonadal anchor cell and acts through LET-23 (transmembrane protein tyrosine kinase and ortholog of EGF receptor) to signal the vulval precursor cells to generate vulval tissue. lin-3 is expressed in the vulval cells later, and recent evidence raises the possibility that lin-3 acts in the vulval cells as a relay signal during vulval induction. It is thus of interest to test the site of action of lin-3 by mosaic analysis. RESULTS: We visualized transgenes in living C. elegans by targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific 256 sequence repeat of the lac operator (lacO) incorporated into transgenes. We engineered animals to express a nuclear-localized GFP-LacI fusion protein. C. elegans cells having a lacO transgene result in nuclear-localized bright spots (i.e., GFP-LacI bound to lacO). Cells with diffuse nuclear fluorescence correspond to unbound nuclear localized GFP-LacI. We detected chromosomes in living animals by chromosomally integrating the array of the lacO repeat sequence and visualizing the integrated transgene with GFP-LacI. This detection system can be applied to determine polyploidy as well as investigating chromosome segregation. To assess the GFP-LacI•lacO system as a marker for mosaic analysis, we conducted genetic mosaic analysis of the epidermal growth factor lin-3, expressed in the anchor cell. We establish that lin-3 acts in the anchor cell to induce vulva development, demonstrating this method's utility in detecting the presence of a transgene. CONCLUSION: The GFP-LacI•lacO transgene detection system works in C. elegans for visualization of chromosomes and extrachromosomal transgenes. It can be used as a marker for genetic mosaic analysis. The lacO repeat sequence as an extrachromosomal array becomes a valuable technique allowing rapid, accurate determination of spontaneous loss of the array, thereby allowing high-resolution mosaic analysis. The lin-3 gene is required in the anchor cell to induce the epidermal vulval precursors cells to undergo vulval development

Coordinated Regulation of Intestinal Functions in C. elegans by LIN-35/Rb and SLR-2

LIN-35 is the sole C. elegans representative of the pocket protein family, which includes the mammalian Retinoblastoma protein pRb and its paralogs p107 and p130. In addition to having a well-established and central role in cell cycle regulation, pocket proteins have been increasingly implicated in the control of critical and diverse developmental and cellular processes. To gain a greater understanding of the roles of pocket proteins during development, we have characterized a synthetic genetic interaction between lin-35 and slr-2, which we show encodes a C2H2-type Zn-finger protein. Whereas animals harboring single mutations in lin-35 or slr-2 are viable and fertile, lin-35; slr-2 double mutants arrest uniformly in early larval development without obvious morphological defects. Using a combination of approaches including transcriptome profiling, mosaic analysis, starvation assays, and expression analysis, we demonstrate that both LIN-35 and SLR-2 act in the intestine to regulate the expression of many genes required for normal nutrient utilization. These findings represent a novel role for pRb family members in the maintenance of organ function. Our studies also shed light on the mechanistic basis of genetic redundancy among transcriptional regulators and suggest that synthetic interactions may result from the synergistic misregulation of one or more common targets

Regulation of Neuronal APL-1 Expression by Cholesterol Starvation

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of b-amyloid plaques composed primarily of the amyloid-b peptide, a cleavage product of amyloid precursor protein (APP). While mutations in APP lead to the development of Familial Alzheimer’s Disease (FAD), sporadic AD has only one clear genetic modifier: the e4 allele of the apolipoprotein E (ApoE) gene. Cholesterol starvation in Caenorhabditis elegans leads to molting and arrest phenotypes similar to loss-of-function mutants of the APP ortholog, apl-1 (amyloid precursor-like protein 1), and lrp-1 (lipoprotein receptor-related protein 1), suggesting a potential interaction between apl-1 and cholesterol metabolism. Methodology/Principal Findings: Previously, we found that RNAi knock-down of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. Here we find the same defect is recapitulated during lrp-1 knock-down and by cholesterol starvation. A cholesterol-free diet or loss of lrp-1 directly affects APL-1 levels as both lead to loss of APL-1::GFP fluorescence in neurons. However, loss of cholesterol does not affect global transcription or protein levels as seen by qPCR and Western blot. Conclusions: Our results show that cholesterol and lrp-1 are involved in the regulation of synaptic transmission, similar to apl-1. Both are able to modulate APL-1 protein levels in neurons, however cholesterol changes do not affect global apl-1 transcription or APL-1 protein indicating the changes are specific to neurons. Thus, regulation of synaptic transmission an

Haul-Out Behavior of Harbor Seals (Phoca vitulina) in Hood Canal, Washington

The goal of this study was to model haul-out behavior of harbor seals (Phoca vitulina) in the Hood Canal region of Washington State with respect to changes in physiological, environmental, and temporal covariates. Previous research has provided a solid understanding of seal haul-out behavior. Here, we expand on that work using a generalized linear mixed model (GLMM) with temporal autocorrelation and a large dataset. Our dataset included behavioral haul-out records from archival and VHF radio tag deployments on 25 individual seals representing 61,430 seal hours. A novel application for increased computational efficiency allowed us to examine this large dataset with a GLMM that appropriately accounts for temporal autocorellation. We found significant relationships with the covariates hour of day, day of year, minutes from high tide and year. Additionally, there was a significant effect of the interaction term hour of day : day of year. This interaction term demonstrated that seals are more likely to haul out during nighttime hours in August and September, but then switch to predominantly daylight haul-out patterns in October and November. We attribute this change in behavior to an effect of human disturbance levels. This study also examined a unique ecological event to determine the role of increased killer whale (Orcinus orca) predation on haul-out behavior. In 2003 and 2005 these harbor seals were exposed to unprecedented levels of killer whale predation and results show an overall increase in haul-out probability after exposure to killer whales. The outcome of this study will be integral to understanding any changes in population abundance as a result of increased killer whale predation

P-Type ATPase TAT-2 Negatively Regulates Monomethyl Branched-Chain Fatty Acid Mediated Function in Post-Embryonic Growth and Development in C. elegans

Monomethyl branched-chain fatty acids (mmBCFAs) are essential for Caenorhabditis elegans growth and development. To identify factors acting downstream of mmBCFAs for their function in growth regulation, we conducted a genetic screen for suppressors of the L1 arrest that occurs in animals depleted of the 17-carbon mmBCFA C17ISO. Three of the suppressor mutations defined an unexpected player, the P-type ATPase TAT-2, which belongs to the flippase family of proteins that are implicated in mediating phospholipid bilayer asymmetry. We provide evidence that TAT-2, but not other TAT genes, has a specific role in antagonizing the regulatory activity of mmBCFAs in intestinal cells. Interestingly, we found that mutations in tat-2 also suppress the lethality caused by inhibition of the first step in sphingolipid biosynthesis. We further showed that the fatty acid side-chains of glycosylceramides contain 20%–30% mmBCFAs and that this fraction is greatly diminished in the absence of mmBCFA biosynthesis. These results suggest a model in which a C17ISO-containing sphingolipid may mediate the regulatory functions of mmBCFAs and is negatively regulated by TAT-2 in intestinal cells. This work indicates a novel connection between a P-type ATPase and the critical regulatory function of a specific fatty acid

CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals

The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.

BACKGROUND: Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform\u27s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. METHODOLOGY/PRINCIPAL FINDINGS: Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. CONCLUSIONS/SIGNIFICANCE: Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However, prohibiting phosphorylation of candidate 14-3-3-binding sites does not impact localization of the fusogen. Hypodermal membrane fusion activity persists when 14-3-3 expression levels are reduced