The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

Background

The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species.

Methodology/Principal Findings

Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of “white tuna” were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus.

Conclusions/Significance

The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010.

–The question is not what you look at, but what you see Thoreau

     

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/Principal Findings

Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-α and IFN-γ. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/Significance

These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells'' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.     

Thioredoxin increases exocytosis by denitrosylating N-ethylmaleimide-sensitive factor

Exocytosis involves membrane fusion between granules and the plasma membrane. Nitric oxide (NO) inhibits exocytosis by chemically modifying N-ethylmaleimide-sensitive factor (NSF), a key component of the exocytic machinery. However, cells recover the ability to release messenger molecules within hours of exposure to NO through unknown mechanisms. We now identify thioredoxin (TRX1) as a denitrosylase that reverses NO inhibition of exocytosis. Endogenously synthesized NO increases S-nitrosylated NSF levels, but S-nitrosylated NSF levels decrease within 3 h after exposure to NO. We found that NO increases the interaction between TRX1 and NSF, and endogenous TRX1 removes NO from S-nitrosylated NSF. Knockdown of TRX1 increases the level of S-nitrosylated NSF, prolongs the inhibition of exocytosis, and suppresses leukocyte adhesion. Taken together, these data show that TRX1 promotes exocytosis by denitrosylating NSF. Our findings suggest that TRX1 might regulate exocytosis in a variety of physiological settings, such as vascular inflammation, thrombosis, and insulin release.     

Hydrogen peroxide regulation of bovine endothelin-converting enzyme-1

Vascular injury leads to the production of reactive oxygen species (ROS), but the mechanisms by which ROS contribute to vascular pathology are not completely understood. We hypothesized that ROS increase endothelin converting enzyme (ECE-1) expression. We found that glucose oxidase (GO) increases ECE-1 mRNA, protein, and activity in bovine aortic endothelial cells. Catalase abolishes this effect. Glucose oxidase treatment of endothelial cells transactivates the ECE-1 promoter. The ECE-1 promoter element that mediates this response to GO is located between -444 and -216 bp. This region contains a STAT response element, and GO activates STAT-3 binding to this STAT response element. Our data suggest that STAT3 mediates hydrogen peroxide induction of ECE-1 expression.     

Effectiveness of the International Phytosanitary Standard ISPM No. 15 on Reducing Wood Borer Infestation Rates in Wood Packaging Material Entering the United States

Numerous bark- and wood-infesting insects have been introduced to new countries by international trade where some have caused severe environmental and economic damage. Wood packaging material (WPM), such as pallets, is one of the high risk pathways for the introduction of wood pests. International recognition of this risk resulted in adoption of International Standards for Phytosanitary Measures No. 15 (ISPM15) in 2002, which provides treatment standards for WPM used in international trade. ISPM15 was originally developed by members of the International Plant Protection Convention to “practically eliminate” the risk of international transport of most bark and wood pests via WPM. The United States (US) implemented ISPM15 in three phases during 2005–2006. We compared pest interception rates of WPM inspected at US ports before and after US implementation of ISPM15 using the US Department of Agriculture AQIM (Agriculture Quarantine Inspection Monitoring) database. Analyses of records from 2003–2009 indicated that WPM infestation rates declined 36–52% following ISPM15 implementation, with results varying in statistical significance depending on the selected starting parameters. Power analyses of the AQIM data indicated there was at least a 95% chance of detecting a statistically significant reduction in infestation rates if they dropped by 90% post-ISPM15, but the probability fell as the impact of ISPM15 lessened. We discuss several factors that could have reduced the apparent impact of ISPM15 on lowering WPM infestation levels, and suggest ways that ISPM15 could be improved. The paucity of international interception data impeded our ability to conduct more thorough analyses of the impact of ISPM15, and demonstrates the need for well-planned sampling programs before and after implementation of major phytosanitary policies so that their effectiveness can be assessed. We also present summary data for bark- and wood-boring insects intercepted on WPM at US ports during 1984–2008.     

Adenosine and 5'-chloro-5'-deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5'-chloro-5'-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.     

An improved procedure for the simultaneous purification in high yield of peroxisomes,lysosomes, and mitochondria from rat liver

Summary Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.     

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.     

Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.

Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.