A molecular ecological approach to the detection and designation of the etiological agents of a model polymicrobial disease.

The application of the original Koch postulates and the molecular Koch postulates in the definition of the etiological agents of polymicrobial diseases has received little or no attention. In the present study, denaturing gradient gel electrophoresis (DGGE) of oral samples (n = 3) from each of 3 categories of animals (healthy, diseased [gingivitis], and then oxytetracycline-treated) was used and revealed different bacterial community structures in a model polymicrobial disease (gingivitis) and after clinical cure. Potential microbes associated with the disease and belonging to the following families were identified: Fusobacteriaceae, Porphyromonadaceae, Flavobacteriaceae, Alcanivoracaceae, Bacteroidaceae, Xanthomonadaceae, and Neisseriaceae. Liquid chromatography-mass spectrophotometric analysis of culturable anaerobic bacteria culture supernatant revealed 3 major compounds (2-hydroxycaproic acid, phenyllactic acid, and indole acetic acid) that differentiated the healthy and disease groups. Results indicate that different microbial community structures were associated with the healthy and disease oral states. The results demonstrate the potential of DGGE as a tool in the detection and designation of etiological agents of polymicrobial diseases.

Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis.

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.

Re-use of remediated soils for the bioremediation of waste oil sludge.

We investigated the possibility of re-using remediated soils for new bioremediation projects by spiking these soils with waste oil sludge in laboratory based microcosms. The level of Total Petroleum Hydrocarbon (TPH) reduction was high (>80%) in naturally attenuated microcosms and was not significantly improved by biostimulation, bioaugmentation and the combined treatment of bioaugmentation and biostimulation by week 12. This indicated that the observed TPH reduction might have been related to the soil's inherent hydrocarbon-degrading potential. Microbial community analysis (16S rDNA and ITS-based Denaturing Gradient Gel Electrophoresis fingerprints) confirmed the dominance of hydrocarbon degrading genera such as Alcanivorax and Scedosporium. Cluster and Shannon diversity analysis revealed similar but stable bacterial and fungal communities in naturally attenuated and amended microcosms indicating that rapid reduction in TPH may not always be accompanied by changes in soil microbial communities. This study has therefore shown that soils previously used for bioremediation can have an improved hydrocarbon degrading potential which was successfully re-harnessed for new projects. This ability to re-harness this potential is attractive because it substantially reduces operational costs as no additional bioremediation treatments are needed. It can also extend a landfill's lifespan as soils can be re-used again before landfill disposal.

Can by-products in country-made alcohols induce acute pancreatitis?

OBJECTIVES: We previously reported a high incidence of alcohol-related acute pancreatitis (AP) in Goa, India, where country-made alcoholic products are consumed in addition to the commercially available alcoholic products. We aimed to analyze the composition of these country-made alcoholic products consumed by a population with a high incidence of alcohol-related AP. METHODS: Three locally distilled alcoholic products (ethanol content, >20%) regularly consumed by patients developing AP, as determined by responses in a patient questionnaire, were selected. Three commercially available products with comparable ethanol content (rum, whiskey, and brandy) were used for comparison. Representative samples were analyzed using gas chromatography/mass spectrometry. Compound assignments used mass spectral searches of the NIST library (2008). RESULTS: Commercially available rum, whiskey, and brandy used for comparison contained the 2 major constituents, ethanol and water. In addition, the country-made alcoholic products contained a higher level of by-products including long-chain alcohols (eg, butanol, propanol), aldehydes (eg, acetaldehyde), acids (eg, acetic acid), and even traces of methanol. CONCLUSIONS: Country-made alcoholic products contain many compounds in addition to ethanol. Given the high incidence of alcohol-related AP in the population where these products are consumed, further evaluation of their constituents in relation to the induction of pancreatic damage is warranted.

LC-MS investigation of oxidation products of phenolic antioxidants.

Two oxidation systems were examined for the oxidation of three groups of phenolic antioxidants; five cinnamic acids, two benzoic acids, and two phenols characteristic of olive fruits. Periodate oxidation, which is reported to produce products similar to polyphenol oxidase, was contrasted with the reactivity of the Fenton system, an inorganic source of hydroxyl radicals. Reaction products were identified as various quinones, dimers, and aldehydes, but the nature of the products differed between the two oxidation systems. Structure-activity effects were also observed for the different phenols. All cinnamic acids in this study reacted with the Fenton reagent to produce benzaldehydes as the main products, with the exception of 5-caffeoylquinic acid. In contrast, periodate oxidation gave no reaction with some of the cinnamic acids. Quinone formation was observed for the two compounds, caffeic acid and 5-caffeoylquinic acid, possessing o-hydroxy groups. Caffeic acid was unusual in that dimer formation was the main initial product of reaction. Benzoic acids were readily oxidized by both systems, but no identifiable products were isolated. Oleuropein was oxidized by both oxidants used in this study, resulting in quinones in each system, whereas little or no oxidation of tyrosol was observed. This highlights the importance of conjugation between the alkene double bond and the hydroxy group. The results question the validity of many existing methods of testing antioxidant activity.

Identification of compound classes in soil and peat fulvic acids as observed by electrospray ionization tandem mass spectrometry.

Soil and peat fulvic acids obtained from the International Humic Substances Society were fractionated by their solubility in methanol and analyzed by electrospray ionization tandem mass spectrometry. Precursor and product ion experiments produced mass spectra that indicated the presence of benzene, phenol, dihydroxy benzene, furan and thiophene carboxylic acids. Standards were used to substantiate the fragmentation patterns observed in the product ion spectra of the fulvic acid samples. This study makes significant progress into the direct identification of individual compounds in humic substances using a non-degradation technique.

Liquid chromatography-mass spectrometry (LC-MS) investigation of the thiobarbituric acid reactive substances (TBARS) reaction.

The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method for the detection of lipid peroxidation. Malondialdehyde is formed as a result of lipid peroxidation and reacts with thiobarbituric acid to form a pink pigment that has an absorption maximum at 532 nm. Other compounds also react with thiobarbituric acid to form colored species that can interfere with this assay, but little is known about these interfering species. This is the first investigation using LC-MS and MS-MS to study the structures of the pink adduct as well as a common unstable yellow interference compound, which absorbs at 455 nm. Also, the presence of barbituric acid impurities in the thiobarbituric acid reagent was found to produce 1:1:1 thiobarbituric acid/malondialdehyde/barbituric acid and 2:1 barbituric acid/malondialdehyde adducts that absorbed at 513 and 490 nm, respectively, indicating that thiobarbituric acid should be purified before use.

Self-esterification of fulvic acid model compounds in methanolic solvents as observed by electrospray ionization mass spectrometry.

The self-esterification of two fulvic acid model compounds in methanolic solvents was studied by electrospray ionization mass spectrometry (ESI-MS). The strongly acidic tetrahydrofurantetracarboxylic acid rapidly self-esterified to form mono- and dimethyl esters when stored in methanol, even at reduced temperatures. The weakly acidic analogue, cyclopentanetetracarboxylic acid, reacted minimally under the same conditions. The use of 50:50 methanol/water as a solvent reduced self-esterification of the strong acid. However, the presence of water promoted the formation of multiply charged ions in the ESI mass spectra. The use of water and 50:50 acetonitrile/water as solvents eliminated self-esterification but the mass spectra still contained multiply charged ions. This study implies that the use of methanolic solvents with humic substances may compromise analytical data through the formation of methyl esters.