Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR ¹H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.     

Recovery of bioactive compounds from Pinus pinaster wood by consecutive extraction stages

Pinus pinaster wood samples, obtained at different positions of three healthy trees, were subjected to two sequential extractions using an Accelerated Solvent Extraction instrument. The first extraction was carried out with hexane (to remove lipophilic extractives) and the second one with acetone/water (95:5 v/v) to recover bioactive phenolic compounds, the target compounds of this study. The extracted fractions were assayed for total yield and composition. The extracts contained a spectrum of phenolic compounds (simple phenolics, phenolic stilbenes, flavonoids and lignans) and non-phenolic components (juvabiones, resin and fatty acids, steryl esters and triglycerides). The fractionation effects achieved by consecutive extractions and the recovery of bioactive phenolics are discussed.     

Assessing common bottlenose dolphin (Tursiops truncatus) population structure in Mississippi Sound and coastal waters of the north central Gulf of Mexico

1. Population structure of highly mobile marine organisms can be complex and difficult to study, but it is important to understand how populations partition themselves within their environment for accurate assessment of both natural and anthropogenic impacts and successful management. The 2010 Deepwater Horizon oil spill negatively impacted common bottlenose dolphins (Tursiops truncatus) within Mississippi Sound and the surrounding north central Gulf of Mexico (GOMx); however, little was known about their underlying population structure in these waters. Thus, it was unclear how many demographically independent populations were affected by the spill.
2. Common bottlenose dolphin samples were collected throughout inshore waters of Mississippi Sound and coastal waters of the north-central GOMx. Mitochondrial DNA control region sequence data and 19 nuclear microsatellite loci were analysed to determine how many populations are present and characterize their range throughout these waters.
3. Bayesian clustering and migration analyses identified two genetically distinct and demographically independent populations: one predominantly inhabiting Mississippi Sound and adjacent coastal waters, and a second population extending generally from offshore of Mobile Bay, Alabama, east along the Florida Panhandle. Neither of these populations align with the currently delineated management stocks previously used to estimate impacts from the oil spill on common bottlenose dolphins in this portion of the GOMx.
4. These results suggest that revisions may be necessary so that management stocks accurately represent the demographically independent populations present in these waters. Furthermore, better comprehension of underlying population structure will enhance impact assessments on common bottlenose dolphins and provide more appropriate baseline data to support future restoration and conservation objectives.

     

Use of the Single Seed Descent Method in Breeding Safflower (Carthamus tinctorius L.)

The single seed descent (SSD) method of selection has been evaluated in safflower (Carthamus tinctorius L.). The initial population of this study was a BC₂F₁ from a cross between a well adapted and a high oil cultivar. Transgressive segregation for yield and oil content producing lines with considerably higher values than the better parent was observed in BC₂F₄ and BC₂F₅ generations. These results and the great saving of time and effort makes the SSD method a very desirable tool for safflower breeders.     

Assessment of the production of antioxidants from winemaking waste solids

Winemaking waste solids (WS, resulting from red grapes after fermentation and distillation to recover spirits) were subjected to various processing schemes for isolating fractions with antioxidant activity. The liquors entrapped in WS as received were separated by pressing and freeze-dried to yield a fraction with antioxidant activity (measured as DPPH radical scavenging capacity) comparable to those of synthetic antioxidants. A second approach based on the direct processing of raw WS in sulfuric acid medium under fixed operational conditions and further extraction of hydrolysis liquors with ethyl acetate enabled the isolation of a fraction with higher antioxidant ability at an improved yield. The most favorable approach started with a washing stage leading to liquors (which were directly freeze-dried to yield 1.20 g of extract/100 g of oven-dry WS and presented an EC50 of 0.41 g of extract/L) and washed solids, which were dried and subjected to hydrolytic processing (i) with water as a reactive in an autocatalyzed reaction (autohydrolysis) or (ii) with sulfuric acid solutions to give an ethyl acetate-soluble fraction with improved antioxidant properties (EC50 in the range of 0.18-0.40 g/L). Samples from washing liquors and processing of washed solids in aqueous medium were subjected to chromatographic fractionation and analysis to give isolates with remarkable antioxidant activity (with EC50 as low as 0.07 g/L) and to identify their major components.     

Conservation value of dispersed tree cover threatened by pasture management

Trees dispersed in pastures are a prominent feature of many Central American landscapes, particularly in cattle producing regions where farmers retain trees to serve as shade, fodder, timber and firewood. The presence of dispersed trees in pastures is often considered as important for the conservation of biodiversity by providing habitat and enhancing landscape connectivity. However, despite their critical productive and environmental roles, little is known about tree distribution within pastures or how farmers’ management decisions influence the trees themselves and their impact on farm productivity and biodiversity conservation. Here, we present a synthesis of (a) the abundance, composition, and size of dispersed trees in four important cattle producing regions of Costa Rica (Caňas and Río Frío) and Nicaragua (Rivas and Matiguás), based on inventory of 18,669 trees on 1492 ha of pasture, (b) the local knowledge, management and use of trees by cattle farmers, and (c) opportunities for ensuring sustainable management of dispersed trees in pasture-dominated landscapes. Dispersed trees were common in all four landscapes, with mean frequency ranging from 8.0 trees ha⁻¹ in Caňas to 33.4 trees ha⁻¹ in Matiguás. A total of 255 tree species were found in pastures across the four landscapes. The total number of tree species per landscape varied from 72 in Rivas to 101 in Caňas and Rio Frio, with mean species richness per farm ranging from 22.9 in Rio Frio to 45.9 in Matiguás. In all four landscapes, a handful of tree species dominated the pastures, with the ten most abundant species in each landscape accounting for >70% of all trees recorded. Most of these common tree species provide fruits or foliage eaten by cattle, or are important timber or firewood species, and are deliberately retained by farmers for these uses. In all four landscapes, farmers had a detailed knowledge of tree attributes affecting pasture and animal productivity, and influenced tree cover through pasture management activities and occasional tree cutting. Current farm management practices are gradually decreasing the diversity of trees in pastures, and in some cases also tree density, reducing their contribution to farm productivity and biodiversity conservation. To reverse this trend, incentives are required to encourage cattle farmers to retain and enhance tree cover in pastures, through the adoption of pasture management practices that favor the regeneration and persistence of a diverse range of tree species.     

In vitro activity of the essential oil of Cinnamomum zeylanicum and eugenol in peroxynitrite-induced oxidative processes

The essential oil obtained from the bark of Cinnamomum zeylanicum Blume (Lauraceae) and three of its main components, eugenol, (E)-cinnamaldehyde, and linalool (representing 82.5% of the total composition), were tested in two in vitro models of peroxynitrite-induced nitration and lipid peroxidation. The essential oil and eugenol showed very powerful activities, decreasing 3-nitrotyrosine formation with IC50 values of 18.4 microg/mL and 46.7 microM, respectively (reference compound, ascorbic acid, 71.3 microg/mL and 405.0 microM) and also inhibiting the peroxynitrite-induced lipid peroxidation showing an IC50 of 2.0 microg/mL and 13.1 microM, respectively, against 59.0 microg/mL (235.5 microM) of the reference compound Trolox. On the contrary, (E)-cinnamaldehyde and linalool were completely inactive.     

Acylglycerol and fatty acid components of pulp,seed, and whole olive fruit oils. Their use to characterize fruit variety by chemometrics

The contents of triacylglycerols and diacylglycerols in three kinds of olive fruit oils (pulp, seed, and whole fruit) were determined. The fatty acid composition and the quality ratios 1,2-diacylglycerols/1,3-diacylglycerols and 1,2-diacylglycerols/total diacylglycerols were also assessed. Seven major Italian olive varieties were considered. Results of univariate statistical analyses indicated that the above analytical parameters (glyceridic ratios excepted) were effective in discriminating between pulp and seed oils. The seed oil fraction did not determine any change in the glyceridic indices and the acylglycerol or fatty acid composition concerning the whole fruit oil (mixture of pulp and seed oil fractions), the weight (%) of seed ( approximately 2%) being by far lower than the weight (%) of pulp ( approximately 85%) (fruit weight basis). Based on the data of triacylglycerol or fatty acid composition, and using appropriate parametric or nonparametric multivariate statistics, the genetic origins (olive variety) of the three fruit oil kinds were characterized.     

Identification and semiquantitation of monensin sodium in liver tissue

A bioautographic technique for the determination of monensin sodium in animal feed has been applied to chicken and beef liver tissues. The reliable limit of sensitivity is 100 ppb, but 10 ppb can be detected. This technique can be used to semiquantitate monensin in tissues by comparing the zones of inhibition of unknown test samples against monensin standards.