PCR Cloning Combined With DNA Barcoding Enables Partial Identification of Fish Species in a Mixed-Species Product

DNA barcoding is a valuable tool for regulatory identification of fish species; however, it does not perform well when multiple species are present within the same food product. Therefore, the objective of this study was to examine the use of PCR cloning to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). Three subsamples from each fish ball underwent DNA extraction, full DNA barcoding (655 bp), and mini-barcoding (226 bp) of the cytochrome c oxidase subunit 1 (CO1) gene. Subsamples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock) with both full and mini-barcoding. However, only tilapia was detected in fish balls containing multiple species when tested with standard barcoding techniques, reflecting an inaccurate representation of the fish mixture and suggesting species bias. PCR cloning allowed for identification of Pacific cod in 86% of the mixed-species fish balls tested with fullbarcode cloning and 100% of the mixed-species fish ball tested with mini-barcode cloning. However, PCR cloning did not enable the identification of walleye pollock. Standard full barcoding produced more high quality sequences compared to minibarcoding yet failed to accurately detect all species present in the tested fish mixtures. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to overcome the species bias observed in this study

Identification of multiple constitutive and inducible hepatic cytochrome P450 enzymes in market weight swine. Drug Metab. Dispos

ABSTRACT: Constitutive swine enzymes analogous to human/rat cytochrome P450 (CYP) isoforms 1A2, 2A6, 2B1/2/6, 2D6, 2E1, 3A1, and 4A1/3 were detected by Western blot analysis. Swine 2E1 has a molecular weight greater than rat 2E1; swine 2B2 has a molecular weight similar to human 2B6. An induction cocktail containing ␤-naphthoflavone, phenobarbital, and dexamethasone induced immunoreactive homologs of 1A1, 1A2, 2B1, 2B2, 3A1, and 3A2. Although the P450 content was increased by induction, there was no difference in the Soret max . Swine 1A1 has a lower molecular weight than swine 1A2 and rat 1A1. A swine 2B1 homolog was seen after induction, with a molecular weight that was lower than rat 2B1 but higher than swine 2B2. Induction did not augment swine 2B2 levels. The 3A homologs have molecular weights similar to their rodent counterparts. Following induction, swine 3A1 levels increased and were accompanied by the appearance of swine 3A2. Induction had no effect on expression of 2A6, 2B6, 2D6, 2E1, or 4A1/3. Enzyme induction increased the specific activities (nmol/min/mg) of substrates specific for 1A (7 of 7 substrates tested), 2A (2/2), 2B (5/5), 2C (1/3), 2D (3/4), 2E (3/3), 3A (3/5), and 4A (1/1). Although the specific activities of the 2E substrates increased, the turnover number for hydroxylation of chlorzoxazone was unchanged and that of p-nitrophenol and aniline were depressed in induced pigs. These results show that swine CYP isoforms are similar to those identified in human and rodents, but they are also different in many ways

Evaluation of DNA Barcoding Methodologies for the Identification of Fish Species in Cooked Products

DNA barcoding is a powerful sequencing-based tool for the detection of fish species substitution. However, various cooking methods have the potential to reduce the quality and success of DNA sequencing. The objective of this study was to determine the effects of common cooking methods on DNA sequencing results with both full-length (655 bp) and mini-barcodes (208–226 bp), and to determine the optimal methodology to use for species identification of various fish products. Six types of fish (salmon, tuna, scad, pollock, swai and tilapia) were prepared in triplicate using the following methods: uncooked, baked, fried, broiled, acid-cooked, smoked and canned. DNA was extracted from each sample and tested using full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were compared based on quality parameters, success rates, and genetic identifications. SH-E mini-barcoding showed the highest overall success rates (92–94%), followed by full barcoding (90%), and SH-D mini-barcoding (67–90%). Across the individual cooking methods, SH-E mini-barcodes performed as well or better than full barcodes for most samples. The sequencing results were fairly consistent across cooking methods with the exception of canning, which showed marked decreases in sequencing success, quality, and length. Despite the reduced sequence length of mini-barcodes compared to full barcodes, identification of fish species was largely consistent across the methods. Overall, the results of this study show that DNA barcoding is a robust tool for fish species identification, and that mini-barcoding has high potential for use as a complement to full barcoding

RACE Project 2116 TOMQAT

: The TOMQAT project will develop the concept of Dynamic Total Quality Management in the context of IBC networks, and will investigate the resulting performance benefits. The present deliverable analyses the requirements to be fulfilled by Total Quality Management, and suggests an approach to meet such requirements. Keywords: Total Quality Management, QoS parameters, TQM Requirements, TQM Approach TOMQAT Page 2 RACE Project R2116 - TOMQAT (c) l994 by the TOMQAT Consortium. Organizations in the TOMQAT consortium are: ALPHA SYSTEMS ANALYSIS INTEGRATION LTD., ALCATEL ISR SA, CRAY COMMUNICATIONS LTD, GMD-FOKUS, INTRACOM SA, NATIONAL TECHNICAL UNIVERSITY OF ATHENS, TECHNICAL UNIVERSITY BERLIN, TELMAT COMMUNICATIONS, WANDEL & GOLTERMANN TOMQAT Page 3 RACE Project R2116 - TOMQAT Executive Summary The main objective of project TOMQAT is to enhance the Total Quality Management (TQM) concept and adapt/apply it in the context of IBC networks serving Multimedia applications. The present d..

RACE Project 2116 TOMQAT

: The TOMQAT project enchances the concept of Dynamic Total Quality Management in the context of IBC networks, and investigates the resulting performance benefits. The present deliverable analyses the requirements to be fulfilled by Total Quality Management, and suggests an approach to meet such requirements, including QoS parameters to be managed and appropriate management mechanisms. Keywords: Total Quality Management, QoS parameters, TQM Requirements, TQM Approach Deliverable 5 Page 2 RACE Project R2116 - TOMQAT (c) l994 by the TOMQAT Consortium. Organizations in the TOMQAT consortium are: ALPHA SYSTEMS ANALYSIS INTEGRATION LTD., ALCATEL ISR SA, CRAY COMMUNICATIONS LTD, GMD-FOKUS, INTRACOM SA, NATIONAL TECHNICAL UNIVERSITY OF ATHENS, TECHNICAL UNIVERSITY BERLIN, TELMAT COMMUNICATIONS, WANDEL & GOLTERMANN Deliverable 5 Page 3 RACE Project R2116 - TOMQAT xecutive Summary The main objective of project TOMQAT is to enhance the Total Quality Management (TQM) concept and adapt/ap..

Ezetimibe added to statin therapy after acute coronary syndromes

BACKGROUND: Statin therapy reduces low-density lipoprotein (LDL) cholesterol levels and the risk of cardiovascular events, but whether the addition of ezetimibe, a nonstatin drug that reduces intestinal cholesterol absorption, can reduce the rate of cardiovascular events further is not known. METHODS: We conducted a double-blind, randomized trial involving 18,144 patients who had been hospitalized for an acute coronary syndrome within the preceding 10 days and had LDL cholesterol levels of 50 to 100 mg per deciliter (1.3 to 2.6 mmol per liter) if they were receiving lipid-lowering therapy or 50 to 125 mg per deciliter (1.3 to 3.2 mmol per liter) if they were not receiving lipid-lowering therapy. The combination of simvastatin (40 mg) and ezetimibe (10 mg) (simvastatin-ezetimibe) was compared with simvastatin (40 mg) and placebo (simvastatin monotherapy). The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, unstable angina requiring rehospitalization, coronary revascularization ( 6530 days after randomization), or nonfatal stroke. The median follow-up was 6 years. RESULTS: The median time-weighted average LDL cholesterol level during the study was 53.7 mg per deciliter (1.4 mmol per liter) in the simvastatin-ezetimibe group, as compared with 69.5 mg per deciliter (1.8 mmol per liter) in the simvastatin-monotherapy group (P<0.001). The Kaplan-Meier event rate for the primary end point at 7 years was 32.7% in the simvastatin-ezetimibe group, as compared with 34.7% in the simvastatin-monotherapy group (absolute risk difference, 2.0 percentage points; hazard ratio, 0.936; 95% confidence interval, 0.89 to 0.99; P = 0.016). Rates of pre-specified muscle, gallbladder, and hepatic adverse effects and cancer were similar in the two groups. CONCLUSIONS: When added to statin therapy, ezetimibe resulted in incremental lowering of LDL cholesterol levels and improved cardiovascular outcomes. Moreover, lowering LDL cholesterol to levels below previous targets provided additional benefit