Two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (?30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at ?20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.     

Biocatalytic synthesis,structural elucidation,antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435®), in acetone at 45 °C. The synthesized esters have been analyzed by ¹H NMR, ¹³C NMR spectroscopy and evaluated for their antioxidant capacity and tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p ?0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.     

Identification of most spectrally distinguishable phenological stage of invasive Phramites australis in Lake Erie wetlands (Canada) for accurate mapping using multispectral satellite imagery

Phragmites australis (Cav.) Trin. ex Steudel subspecies australis is one of the worst plant invaders in wetlands of North America. Remote sensing is the most cost-effective method to track its spread given its widespread distribution and rapid colonization rate. We hypothesize that the morphological and/or physiological features associated with different phenological states of Phragmites can influence their reflectance signal and thus affect mapping accuracies. We tested this hypothesis by comparing classification accuracies of cloud-free images acquired by Landsat 7, Landsat 8, and Sentinel 2 at roughly monthly intervals over a calendar year for two wetlands in southern Ontario. We used the Support Vector Machines classification and employed field observations and image acquired from unmanned aerial vehicle (8 cm) to perform accuracy assessments. The highest Phragmites producer’s, user’s, and overall accuracy (96.00, 91.11, and 88.56% respectively) were provided by images acquired in late summer and fall period. During this period, green, Near Infrared, and Short-Wave Infrared bands generated more unique reflectance signals for Phragmites. Both Normalized Difference Vegetation Index and Normalized Difference Water Index showed significant difference between Phragmites and the most confused classes (cattail; Typha latifolia L., and meadow marsh) during the late summer and fall period. Since meadow marsh separated out best from Phragmites and cattail in the February image, we used it to mask the meadow marsh in the July image to reduce confusion. The unique reflectance signal of Phragmites in late summer and fall is likely due to prolonged greenness of Phragmites when compared to other wetland vegetation, large, distinct inflorescence, and the water content of Phragmites during this period.

     

Diagnostic method-based underestimation of leptospirosis in clinical and research settings; an experience from a large prospective study in a high endemic setting

BackgroundLeptospirosis has globally significant human mortality and morbidity, yet estimating the clinical and public health burden of leptospirosis is challenging because timely diagnosis remains limited. The goal of the present study was to evaluate leptospirosis undercounting by current standard methods in both clinical and epidemiological study settings.Methodology/Principal findingsA prospective hospital-based study was conducted in multiple hospitals in Sri Lanka from 2016 to 2019. Culture, whole blood, and urine samples were collected from clinically suspected leptospirosis cases and patients with undifferentiated fever. Analysis of biological samples from 1,734 subjects confirmed 591 (34.1%) cases as leptospirosis and 297 (17.1%) were classified as “probable” leptospirosis cases. Whole blood quantitative PCR (qPCR) did identify the most cases (322/540(60%)) but missed 40%. Cases missed by each method include; urine qPCR, 70% (153/220); acute sample microscopic agglutination test (MAT), 80% (409/510); paired serum sample MAT, 58% (98/170); and surveillance clinical case definition, 53% (265/496). qPCR of negative culture samples after six months of observation was of diagnostic value retrospectively with but missed 58% of positives (109/353).ConclusionLeptospirosis disease burden estimates should consider the limitations of standard diagnostic tests. qPCR of multiple sample types should be used as a leading standard test for diagnosing acute leptospirosis.     

Homology modeling of plant cytochrome P450s

Plant P450 monooxygenases represent the largest family of plant proteins and the largest collection of P450s available for comparative studies and biotechnological applications. They have been shown to catalyze a diverse array of difficult chemical reactions and have demonstrated potential to be used in pharmacological, agronomic and phytoremediative applications. Central to our use of these catalytically competent enzymes is the need to understand their interactions with substrates. Because most characterized plant P450s are membrane-bound proteins that are resistant to standard X-ray and NMR structure determinations, homology modeling represents a reliable and relatively rapid alternative method for analyzing structure-function relationships and predicting substrates for many P450s that are only now being characterized. These methods, which are being widely used in mammalian P450 structure-function studies, can allow plant biologists to define critical residues interacting with substrates and, in a directed fashion, alter the reactivities of individual monooxygenases. The homology modelings that have been done on a limited number of plant P450s and the site-directed mutations that validate them indicate that current modeling and substrate docking procedures are capable of providing structural explanations for sequence variants as well as for predicting functional characteristics of undefined P450s.     

The effects of organic amendments on mineral element uptake and fruit quality of raspberries

The organic produce industry is gaining popularity with consumers because of the perception for healthier foods and the environmental benefits of this agricultural practice. Common amendments in organic agriculture include compost and compost tea, the latter being a relatively new product in North America. The main objective of this study was to assess the effectiveness of ruminant and municipal solid waste compost and compost teas made from these composts on selected soil, leaf and fruit parameters of raspberries. Generally, foliar compost tea application was as effective as compost addition in raspberry production. Concentrations of K in leaf and fruits were significantly lower (p?=?0.05) in compost tea treated raspberries. Furthermore, the compost tea increased leaf Na compared to compost amendments which suggested that raspberries preferentially take up Na via foliar applications compared to root Na applications. The yield, total antioxidant capacity of fruit, and vitamin C content of fruit were not affected by treatment but differed greatly among years. Precipitation also varied greatly among years as did soil nutrient concentrations and may have influenced yield, total antioxidant capacity, and vitamin C content.     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Variation in lipid classes and fatty acid composition of salmon shark (Lamna ditropis) liver with season and gender

The influence of season and gender on lipid content, lipid classes, and fatty acid compositions was assessed in livers of salmon shark (Lamna ditropis), caught in the Pacific Ocean. No significant difference in the hepatosomatic index was noted with season, though the lipid content was significantly higher (P<0.05) in winter. Triacylglycerol (TAG) was identified as the predominant lipid class (78.5-82.0%), followed by sterol esters (5.7-9.1%) and hydrocarbons (3.4-5.4%). No significant differences were observed in TAG composition with respect to the season or gender. However, diacylglyceryl ether contents were significantly higher (P<0.05) in winter (3.8-5.3%) than those obtained in summer (1.3-1.1%). Polyunsaturated fatty acids constituted the major fatty acid class of salmon shark total liver lipid and docosahexaenoic acid (C22:6n-3) (22.7-28.4%) was the most abundant fatty acid which was significantly lower (P<0.05) in winter. These results suggested that lipid characteristics of salmon shark liver were influenced by season, but not by gender.