Metabolic footprinting as a tool for discriminating between brewing yeasts.

Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements. Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler). Culture supernatants were taken and analyzed for their "metabolic footprints" by using direct-injection mass spectrometry. Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action. Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses. Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances.

In order to develop convenient and reproducible methods for the identification of Ginseng drugs at a DNA level, PCR-Restriction fragment length polymorphism (PCR-RFLP) and Mutant allele specific amplification (MASA) analyses were applied, based on differences of the 18S rRNA gene sequence among three Panax species. The PCR product of each species on the 18S rRNA gene was digested with the restriction enzymes Ban II and Dde I. Each fragment gave unique electrophoretic profiles for each species (PCR-RFLP analysis). The extracted DNA of each species was amplified by PCR using a designed species-specific oligonucleotide primer. The expected size of the fragments corresponding to each species were detected only when the optimum temperature and reaction time for annealing and extension were established (MASA analysis). These two analytical methods were carried out on three Ginseng drugs and the same results as in their original plants were obtained. The results suggest that PCR-RFLP and MASA analyses under the established conditions are convenient for identifying three Ginseng drugs. Moreover, to insure completion of the identification, a partial sequence of the plastid gene matK was determined in addition to the 18S rRNA gene. The gene sequences of three Panax species were of 1259 base pairs and that of P. quinquefolius was different from the other two at nucleotide position 102.

Introduction. Colorectal cancer (CRC) is the second leading cause of cancer related deaths, due to the development of distant metastases in many CRC patients. To detect metastatic disease in early stage, the identification of tumor cells circulating in the peripheral blood of patients (CTCs) is of outmost importance. Several techniques have recently been developed which can detect CTCs, two of them are the CellSearch® system and the EpiSpot assay. We have investigated genomic aberrations and gene expression patterns in primary colorectal tumors in correlation with CTC status of patients. This may lead to the identification of the underlying mechanisms of metastatic spread and potential new markers for detecting the tumor's capability to disseminate. Methods. Peripheral blood of 48 colorectal patients was investigated by CellSearch® and EpiSpot for CTCs. Samples with at least one CTC, detected with either the CellSearch® system or the EpiSpot assay were considered as positive. Genomic DNA and mRNA extracted from the primary tumors of the same patients were investigated with Illumina based Comparative Genomic Hybridization (CGH) and Gene Expression profiling (GEP) microarrays. CGH arrays covered the whole genome with an average resolution of 5kb and GEP arrays covered more than 25,000 annotated genes. Results. Twenty-nine patients were diagnosed to be positive for CTCs, either with the EpiSpot assay or the CellSearch® system; negative for CTCs in both assays were 17 blood samples. Array-CGH showed that primary tumors from CTC positive patients harbored significantly more often loss of chromosome 15q11 (Fisher's exact test, p<0.01). The Significance Analysis of Microarrays (SAM) revealed 13 genes to be differentially expressed between both groups. These genes are mainly involved in kinase activity and ATP-binding, and are located in the plasma membrane. Furthermore, CYFIP1 was downregulated in colorectal tumors from CTC positive patients compared with CTC negative patients which correlated with its loss detected with array-CGH. CYFIP1 is a known putative invasion suppressor gene in epithelial cancers. Conclusion. Our results suggest that several molecular and genomic profiles can be identified as marker for tumor cell dissemination in CRC. More over, significant correlation between CTC status and the primary tumor's genomic profile indicates a biological rationale for tumor cell dissemination rather than a random effect or shedding of cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2165.

Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.

Metabolic footprinting has been applied as a non-invasive approach to study the behaviour and responses of cultured cells to a range of genetic and environmental perturbations. Gas chromatography interfaced with time-of-flight mass spectrometry (GC-ToF-MS) has become a powerful tool for the analysis of metabolome-derived samples. Generally, two data analysis strategies are used to interrogate and understand the biological patterns within the multi-dimensional data. The first strategy, a commoner one, uses multivariate analysis after chromatographic and mass spectral deconvolution, and the second strategy directly employs multivariate analysis of non-deconvoluted data. Here, two strategies have been assessed for the separation and classification of metabolic footprints (exometabolomes) of two strains of Candida albicans grown on three different carbon sources (glycerol, glucose and galactose). We describe a semi-automated approach that simultaneously processes all samples using the chromatographic dimension data with principal components analysis (PCA), which can include data pre-processing before PCA analysis. The preprocessed and non-deconvoluted total ion chromatogram (TIC) data showed good separation of classes defined by growth on different carbon sources and when comparing the two strains grown on the same carbon source separation was achieved for strains grown on glucose and glycerol after preprocessing. The discrimination observed is greater for preprocessed and non-deconvoluted TIC data than for that of preprocessed and non-deconvoluted single ion chromatogram data. The results from the proposed approach with those produced by MZmine were compared. The results from MZmine data depicted separations in PCA space according to carbon source, but no separation was seen when studying strains grown on the same carbon source. Our research showed that the non-deconvoluted strategy is suitable for fast comparison of large sets of GC-MS data although it will not directly provide biological information. The non-deconvoluted strategy can avoid problems of analyzing complex samples using deconvolution software.

In Thailand many species of Stemona were reported and biological activities such as insecticide, insect repellency and anti-tumor were different. Thus, accurate identification of Stemona is needed in order to ensure their efficacies. The identification based on morphological characters of each species alone is difficult because the morphology of Stemona is similar and they are often sold as crude drug which lose their original feature. The purpose of this study was to analyze DNA fingerprint of six Stemona in Thailand; S. tuberosa Lour., S. collinsae Craib, S. phyllantha Gagnep., S. burkillii Prain, S. aphylla craib and Stemona sp., and developed PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) as a genetic marker in order to use as a convenient tool for identification. The nucleotide sequences of three DNA regions; matK, trnH-psbA and ITS1, were exploited to identify these six Stemona species in Thailand. A result of the comparison of partial matK could be classified Stemona into two groups, S. tuberosa group and S. collinsae group, concerning with their morphology and chemical composition. The nucleotide sequences of trnH-psbA and ITS1 could be used to discriminate six Stemona spcies in Thailand. On the basis of difference among the partial matK gene and ITS1 region, the PCR-RFLP analysis was performed. The restriction patterns showed distinct and polymorphic fingerprints among Stemona spp. and were able to apply for crude drug identification. These results exhibited that the obtained nucleotide sequences could be used to identify Stemona in Thailand. PCR-RFLP genetic marker developed here could be used as a convenient tool for authentication of Stemona and also be applied to their commercial crude drugs

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.

Annual consumption of amantadine increased abruptly after its approval for the treatment of influenza A virus infections in Japan in 1998, and the emergence of amantadine-resistant viruses is now a matter of concern. To detect resistant influenza A virus strains, we have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for nasopharyngeal swabs. Three different primer sets for nested PCR were designed to incorporate restriction sites into the amplicon to differentiate single-amino-acid substitutions at positions 27, 30, and 31 that confer resistance in the transmembrane domain of the M2 protein. Each PCR product was digested with respective endonucleases (BspLU11I for amino acid change at position 27, HhaI for position 30, and ScaI for position 31), and the polymorphisms were determined by electrophoresis. Thirty-four (24.1%) of 141 PCR-positive samples had resistance patterns in eight nursing homes in the 1998-1999 season. Thirty-one viruses (91.2%) showed a change at position 31 (serine to asparagine), three viruses (8.8%) showed a change at position 30 (alanine to threonine), and none showed a change at position 27. The incidence of resistant viruses did not show any significant difference between four facilities where amantadine was used mainly for influenza treatment and four other facilities where it was used only for Parkinson's disease, values being 27.6 and 16.3%, respectively. We have confirmed that the PCR-RFLP method is useful for detecting amantadine-resistant strains directly from nasopharyngeal swabs and that resistant viruses were circulating in nursing homes where the drug was used not only for influenza virus but also for Parkinson's disease.

The phylogeny of 12 Campylobacter species and reference strains of Arcobacter butzleri and Helicobacter pylori was studied based on partial 593-bp groEL gene sequences. The topology of the phylogenetic neighbor-joining tree based on the groEL gene was similar to that of the tree based on the 16S rRNA gene. However, groEL was found to provide a better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared with those for the 16S rRNA gene (range, 90 to 99%) and high intraspecies sequence similarities (range, 95 to 100%; average, 99%). A new universal reverse primer that amplifies a 517-bp fragment of the groEL gene was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of 68 strains representing 11 Campylobacter species as well as reference strains of A. butzlerii and H. pylori. Digestion with the AluI enzyme discriminated all Campylobacter species included in the study but showed more intraspecies diversity than digestion with the ApoI enzyme. A hippurate-negative variant of Campylobacter jejuni with a high level of groEL sequence similarity to both C. jejuni (96%) and C. coli (94%) gave a unique AluI profile and an ApoI profile identical to those of other C. jejuni strains. In conclusion, groEL gene sequencing and PCR-RFLP analysis are recommended as valuable tools for the identification of Campylobacter species.

Differentiation of non-aureus staphylococci species isolated from bovine mastitis by PCR-RFLP of groEL and gap genes in comparison to MALDI-TOF mass spectrometry

BackgroundThe largemouth bass (Micropterus salmoides), an economically important freshwater fish species widely farmed in China, is traditionally cultured using a diet of forage fish. However, given the global decline in forage fish fisheries and increasing rates of waterbody pollution and disease outbreaks during traditional culturing, there is a growing trend of replacing forage fish with formulated feed in the largemouth bass breeding industry. The specific molecular mechanisms associated with such dietary transition in this fish are, nevertheless, poorly understood.MethodsTo identify single nucleotide polymorphisms (SNPs) related to food habit domestication traits and growth traits in largemouth bass fry, we initially genotyped fry using eight candidate SNPs based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, with genetic parameters being determined using Popgen32 and Cervus 3.0. Subsequently, we assessed the associations between food habit domestication traits of largemouth bass fry and these SNPs using the Chi-square test or Fisher’s exact test. Furthermore, we used a general linear model to assess the relationships between the growth traits of largemouth bass fry and these SNPs. The Pearson correlation coefficient between growth traits and the SNPs was also determined using bivariate correlation analysis in IBM SPSS Statistics 22. Finally, the phenotypic variation explained (PVE) by the SNPs was calculated by regression analysis in Microsoft Excel.ResultsThe genotyping results obtained based on PCR-RFLP analysis were consistent with those of direct sequencing. Five SNPs (SNP01, SNP02, SNP04, SNP05, and SNP06) were found to be significantly correlated with the food habit domestication traits of fry (P < 0.05); SNP01 (P = 0.0011) and SNP04 (P = 0.0055) particularly, had showed highly significant associations. With respect to growth traits, we detected significant correlations with the two SNPs (SNP01 and SNP07) (P < 0.05), with SNP01 being significantly correlated with body length, and height (P < 0.05), and SNP07 being significantly correlated with body height only (P < 0.05).ConclusionsOur findings indicated that the PCR-RFLP can be used as a low-cost genotyping method to identify SNPs related to food habit domestication and growth traits in largemouth bass, and that these trait-related SNPs might provide a molecular basis for the future breeding of new varieties of largemouth bass.

Chromosomal abnormalities (CAs) can cause spontaneous miscarriage and increase the incidence of subsequent pregnancy loss and other complications. Presently, CAs are detected mainly by array comparative genomic hybridization (CGH) and single nucleotide polymorphism microarrays. The present study developed a low-coverage next-generation sequencing method to detect CAs in spontaneous miscarriage and assess its clinical performance. In total, 1,401 patients who had experienced an abortion were enrolled in the present study and divided into two groups. In group I, 437 samples that had been previously validated by array CGH were used to establish a method to detect CAs using a semiconductor sequencing platform. In group II, 964 samples, which were not verified, were assessed using established methods with respect to clinical significance. Copy number variant (CNV)-positive and euploidy samples were verified by array CGH and short tandem repeat profiling, respectively, based on quantitative fluorescent PCR. The low-coverage sequencing method detected CNVs >1 Mb in length and a total of 3.5 million unique reads. Similar results to array CGH were obtained in group I, except for six CNVs <1 Mb long. In group II, there were 341 aneuploidies, 195 CNVs, 25 mosaicisms and 403 euploidies. Overall, among the 1,401 abortion samples, there were 536 aneuploidies, 263 CNVs, 34 mosaicisms, and 568 euploidies. Trisomies were present in all autosomal chromosomes. The most common aneuploidies were T16, monosomy X, T22, T15, T21 and T13. Furthermore, one tetrasomy 21, one CNV associated with Wolf-Hirschhorn syndrome, one associated with DiGeorge syndrome and one associated with both Prader-Willi and Angelman syndromes were identified. These four cases were confirmed by short tandem repeat profiling and array CGH. Quantitative fluorescent PCR revealed nine polyploidy samples. The present method demonstrated equivalent efficacy to that of array CGH in detecting CNVs >1 Mb, with advantages of requiring less input DNA and lower cost.

Burkitt's lymphoma is an aggressive B-cell lymphoma characterized by typical morphological, immunophenotypic and molecular features. Gene expression profiling provided a molecular signature of Burkitt's lymphoma, but also demonstrated that a subset of aggressive B-cell lymphomas not fulfilling the current World Health Organization criteria for the diagnosis of Burkitt's lymphoma nonetheless show a molecular signature of Burkitt's lymphoma ('discrepant Burkitt's lymphoma'). Given the different treatment of Burkitt's lymphoma and diffuse large B-cell lymphomas we investigated molecular differences within gene expression-defined Burkitt's lymphoma. We studied tumors from 51 Burkitt's lymphoma patients, comprising 26 with classic Burkitt's lymphoma, 17 with atypical Burkitt's lymphoma and 8 with 'discrepant Burkitt's lymphoma', by comparative genomic hybridization and gene expression profiling. Classic and atypical Burkitt's lymphoma (excluding 'discrepant Burkitt's lymphoma'), in adult and pediatric cases do not differ in underlying genomic imbalances or gene expression suggesting that these subgroups are molecularly homogeneous. 'Discrepant Burkitt's lymphoma', however, differ dramatically in the absolute number of alterations from classic/atypical Burkitt's lymphoma and from diffuse large B-cell lymphoma. Moreover, this category includes lymphomas that carry both the t(14;18) and t(8;14) translocations and are clinically characterized by presentation in adult patients and an aggressive course. Pediatric and adult Burkitt's lymphoma are molecularly homogeneous, whereas 'discrepant Burkitt's lymphoma' differ in underlying genetic and clinical features from typical/atypical Burkitt's lymphoma. 'Discrepant Burkitt's lymphoma' may therefore form a distinct genetic subgroup of aggressive B-cell lymphomas, which show poor response to multi-agent chemotherapy.

PCR-based multi-locus DNA fingerprints represent one of the most informative and cost-effective measures of genetic diversity and are useful population-level biomarkers of toxicologic and other anthropogenic impacts. However, concerns about reproducibility of DNA fingerprints have limited their wider use in environmental biology. We assessed polymorphism and reproducibility of two common fingerprinting techniques, RAPD (randomly amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), in pedigreed populations of rainbow trout (Oncorhynchus mykiss) to derive general rules for selective removal of problematic fingerprint bands. We found that by excluding bands that comprised less than 1% of total intensity, and by excluding the largest and smallest 10% of the bands, we could achieve nearly 100% reproducibility of AFLP fingerprints. Similar application of band exclusion criteria to RAPD fingerprints did not significantly enhance their reproducibility, and at least 15% of RAPD bands were not fully repeatable, heritable, or transmittable. The RAPD technique produced more polymorphic fingerprints than AFLP; however, considering that a substantial proportion of RAPD markers did not demonstrate Mendelian inheritance patterns, the AFLP methodology is to be preferred for future research.