Eco-Efficient Quantification of Glucosinolates in Camelina Seed, Oil, and Defatted Meal: Optimization, Development, and Validation of a UPLC-DAD Method.

Camelina sativa (camelina) seed, oil, and defatted meal are widely used for food, animal feed, and other purposes. The accurate quantification of camelina glucosinolates is critical as their functionalities are highly dose-dependent. The classic quantification of glucosinolates in camelina products involves tedious desulfation steps, toxic reagents, and a lengthy instrument time because glucosinolates are easy to degrade and subject to interference in the liquid chromatography. Thus, we developed and validated an eco-efficient UPLC-DAD method for determining glucoarabin (GS9), glucocamelinin (GS10), and homoglucocamelinin (GS11) in camelina seed, oil, and defatted meal. Glucosinolates were extracted using 80% cold methanol to denature myrosinase, and were separated by an HSS T3 column without desulfation. Glucotropaeolin was used as an internal standard to track analyte degradation and loss during sample preparation. The method has shown high precision (relative standard deviations ranging from 4.12% to 6.54%) and accuracy (>94.4% spike recovery) for GS9-11, and all validation parameters passed the industry-consensus AOAC Appendix F criteria. To our best knowledge, this is the first eco-efficient and low-cost analytical method that is validated against strict AOAC criteria for the quantification of intact camelina glucosinolates. The method is suitable to be adopted as a new industrial testing standard to assist in the quality control of camelina products.

Polymerase chain reaction-based species verification and microsatellite analysis for canine cell line validation.

Cell line cross-contamination as well as genetic drift during passaging have been acknowledged as widespread problems since the 1960s. Improper cell line identification can invalidate results and, if not discovered, pollute the scientific community's body of knowledge with regard to cancer cell lines, their gene expression, and their drug susceptibilities. Despite the obvious need, validation of cell line identity is not yet widely required, and the problem persists. A highly sensitive polymerase chain reaction (PCR)-based approach and short tandem repeat (STR) profiling were used to examine the prevalence of inter- and intraspecies cell line contamination in a veterinary research setting. First, 60 cell lines from 6 laboratories were tested with multiplex species-specific PCR capable of identifying 6 commonly used species. Of these, 3 were determined to be misidentified by species. Second, to identify intraspecies contamination among canine cancer cell lines, 29 canine lines from 3 different laboratories were analyzed with STR fingerprinting. Using this methodology, 3 canine cell lines were determined to be misidentified or cross-contaminated by other canine cell lines. Finally, genetic drift was observed within 1 cell line obtained from different laboratories. These findings emphasize the importance of cell line validation as a critical component of "good cell culture practice." A database of the STR profiles obtained in the current study has been established for future comparison and validation of canine cell lines by investigators at Colorado State University and other institutions.

DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA).

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.