Retention time prediction and protein identification

Proteins are commonly identified through enzymatic digestion and generation of short sequence tags or fingerprints of peptide masses by mass spectrometry. Separation methods, such as liquid chromatography and electrophoresis, are often used to fractionate complex protein or peptide mixtures and these separations also provide information on the different species, such as molecular weight and isoelectric point from electrophoresis and hydrophobicity in reversed-phase chromatography. These are also properties that can be predicted from amino acid sequences derived from genomic sequences and used in protein identification. This chapter reviews recently introduced methods based on retention time prediction to extract information from chromatographic separations and the applications to protein identification in organisms with small and large genomes. Novel data on retention time prediction of posttranslationally modified peptides is also presented.

Diet digestibility, rate of passage, and eating and rumination behavior of Jersey and Holstein cows

Diet digestibility and rate of passage, eating and rumination behavior, dry matter intake (DMI), and lactation performance were compared in 6 Jersey and 6 Holstein multiparous cows. Cows were fed gestation diets according to body weight (BW) beginning 7 wk before expected calving and ad libitum amounts of a lactation diet postpartum. Diet digestibility and rate of passage were measured in 5-d periods at wk 5 prepartum and wk 6 and 14 of lactation. Eating and ruminating behavior was measured over 5-d periods at wk 5 and 2 prepartum and wk 2, 6, 10, and 14 of lactation. Milk yield and DMI were higher in Holsteins, but milk energy output per kilogram of metabolic BW (BW0.75) and intake capacity (DMI/kg of BW) did not differ between breeds. Holsteins spent longer ruminating per day compared with Jerseys, but daily eating time did not differ between breeds. Jerseys spent more time eating and ruminating per unit of ingested feed. The duration and number of meals consumed did not differ between breeds, but the meals consumed by Jerseys were distributed more evenly throughout each 24-h period, providing a more regular supply of feed to the rumen. Feed passed through the digestive tract more quickly in Jerseys compared with Holsteins, suggesting particle breakdown and rumen outflow were faster in Jerseys, but this may also reflect the relative size of their digestive tract. Neutral detergent fiber digestibility was greater in Jerseys, despite the shorter rumen retention time, but digestibility of dry matter, organic matter, starch, and N did not differ between breeds. Utilization of digested N for tissue retention was higher at wk 5 prepartum and lower at wk 14 of lactation in Jerseys. In contrast to numerous published studies, intake capacity of Jerseys was not higher than that of Holsteins, but in the present study, cows were selected on the basis of equal expected milk energy yield per kilogram of metabolic BW. Digestibility of neutral detergent fiber and rate of digesta passage were higher in Jerseys, probably as a consequence of increased mastication per unit of feed consumed in Jerseys and their smaller size.

Effect of method of application of a fibrolytic enzyme product on digestive processes and milk production in Holstein-Friesian cows

Four multiparous cows with cannulas in the rumen and proximal duodenum were used in early lactation in a 4 x 4 Latin square experiment to investigate the effect of method of application of a fibrolytic enzyme product on digestive processes and milk production. The cows were given ad libitum a total mixed ration (TMR) composed of 57% (dry matter basis) forage (3:1 corn silage:grass silage) and 43% concentrates. The TMR contained (g/kg dry matter): 274 neutral detergent fiber, 295 starch, 180 crude protein. Treatments were TMR alone or TMR with the enzyme product added (2 kg/1000 kg TMR dry matter) either sprayed on the TMR 1 h before the morning feed (TMR-E), sprayed only on the concentrate the day before feeding (Concs-E), or infused into the rumen for 14 h/d (Rumen-E). There Was no significant effect on either feed intake or milk yield but both were highest on TMR-E. Rumen digestibility of dry matter, organic matter, and starch was unaffected by the enzyme. Digestibility of NDF was lowest on TMR-E in the rumen but highest postruminally. Total Tract digestibility was highest on TMR-E for dry matter, organic matter, and starch but treatment differences were nonsignificant for neutral detergent fiber: Corn silage stover retention time in the rumen was reduced by all enzyme treatments but postruminal transit time vas increased so the decline in total tract retention. time with enzymes was not significant. It is suggested that the tendency for enzymes to reduce particle retention time in the rumen may, by reducing the time available for fibrolysis to occur, at least partly explain the variability in the reported responses to enzyme treatment.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as N-15. By utilising hydroponic media that contain N-15 inorganic salts as the sole nitrogen source, near to 100% N-15-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled N-14- and N-15-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of N-14/N-15 peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the N-14 and N-15 peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct N-14 and N-15 peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). (C) 2008 Elsevier Ltd. All rights reserved.

Modelling fixed plant and algal dynamics in rivers: an application to the River Frome

The development of eutrophication in river systems is poorly understood given the complex relationship between fixed plants, algae, hydrodynamics, water chemistry and solar radiation. However there is a pressing need to understand the relationship between the ecological status of rivers and the controlling environmental factors to help the reasoned implementation of the Water Framework Directive and Catchment Sensitive Farming in the UK. This research aims to create a dynamic, process-based, mathematical in-stream model to simulate the growth and competition of different vegetation types (macrophytes, phytoplankton and benthic algae) in rivers. The model, applied to the River Frome (Dorset, UK), captured well the seasonality of simulated vegetation types (suspended algae, macrophytes, epiphytes, sediment biofilm). Macrophyte results showed that local knowledge is important for explaining unusual changes in biomass. Fixed algae simulations indicated the need for the more detailed representation of various herbivorous grazer groups, however this would increase the model complexity, the number of model parameters and the required observation data to better define the model. The model results also highlighted that simulating only phytoplankton is insufficient in river systems, because the majority of the suspended algae have benthic origin in short retention time rivers. Therefore, there is a need for modelling tools that link the benthic and free-floating habitats.

Effect of feeding fat or intrajugular infusion of glucagon-like peptide-1 and cholecystokinin on dry matter intake, digestibility, and digesta rate of passage in growing wethers

A cause and effect relationship between glucagon-like peptide 1 (7, 36) amide (GLP-1) and cholecystokinin (CCK) and DMI regulation has not been established in ruminants. Three randomized complete block experiments were conducted to determine the effect of feeding fat or infusing GLP-1 or CCK intravenously on DMI, nutrient digestibility, and Cr rate of passage (using Cr(2)O(3) as a marker) in wethers. A total of 18 Targhee × Hampshire wethers (36.5 ± 2.5 kg of BW) were used, and each experiment consisted of four 21-d periods (14 d for adaptation and 7 d for infusion and sampling). Wethers allotted to the control treatments served as the controls for all 3 experiments; experiments were performed simultaneously. The basal diet was 60% concentrate and 40% forage. In Exp. 1, treatments were the control (0% added fat) and addition of 4 or 6% Ca salts of palm oil fatty acids (DM basis). Treatments in Exp. 2 and 3 were the control and 3 jugular vein infusion dosages of GLP-1 (0.052, 0.103, or 0.155 µg•kg of BW(-1)•d(-1)) or CCK (0.069, 0.138, or 0.207 µg•kg of BW(-1)•d(-1)), respectively. Increases in plasma GLP-1 and CCK concentrations during hormone infusions were comparable with increases observed when increasing amounts of fat were fed. Feeding fat and infusion of GLP-1 tended (linear, P = 0.12; quadratic, P = 0.13) to decrease DMI. Infusion of CCK did not affect (P > 0.21) DMI. Retention time of Cr in the total gastrointestinal tract decreased (linear, P < 0.01) when fat was fed, but was not affected by GLP-1 or CCK infusion. In conclusion, jugular vein infusion produced similar plasma CCK and GLP-1 concentrations as observed when fat was fed. The effects of feeding fat on DMI may be partially regulated by plasma concentration of GLP-1, but are not likely due solely to changes in a single hormone concentration.

Multiwalled nanotubes formed by catanionic mixtures of drug amphiphiles

Mixing of oppositely charged amphiphilic molecules (catanionic mixing) offers an attractive strategy to produce morphologies different from those formed by individual molecules. We report here on the use of catanionic mixing of anticancer drug amphiphiles to construct multiwalled nanotubes containing a fixed and high drug loading. We found that the molecular mixing ratio, the solvent composition, the overall drug concentrations, as well as the molecular design of the studied amphiphiles are all important experimental parameters contributing to the tubular morphology. We believe these results demonstrate the remarkable potential that anticancer drugs could offer to self-assemble into discrete nanostructures and also provide important insight into the formation mechanism of nanotubes by catanionic mixtures. Our preliminary animal studies reveal that the CPT nanotubes show significantly prolonged retention time in the tumor site after intratumoral injection.

Cacao swollen shoot virus in Nigeria: analysis of a pathogen and its vectors

Cacao swollen shoot virus (CSSV) causes the Cacao swollen shoot virus disease (CSSVD) and significantly reduces production in West African cacao. This study characterised the current status of the disease in the major cacao growing States in Nigeria and attempted a clarification on the manner of CSSV transmission. Two separate field surveys and sample collections were conducted in Nigeria in summer 2012 and spring 2013. PCR-based screening of cacao leaf samples and subsequent DNA sequencing showed that the disease continues to persist in Ondo and Oyo States and in new cacao sites in Abia, Akwa Ibom, Cross River and Edo States. Mealybug samples collected were identified using a robust approach involving environmental scanning electron microscopy, histology and DNA barcoding, which highlighted the importance of integrative taxonomy in the study. The results show that the genus Planococcus (Planococcus citri (Risso) and/or Planococcus minor (Maskell)) was the most abundant vector (73.5%) at the sites examined followed by Formicococcus njalensis (Laing) (19.0 %). In a laboratory study, the feeding behaviour of Pl. citri, Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus viburni (Signoret) on cacao were investigated using electrical penetration graph (EPG) analysis. EPG waveforms reflecting intercellular stylet penetration (C), extracellular salivation (E1e), salivation in sieve elements (E1), phloem ingestion (E2), derailed stylet mechanics (F), xylem ingestion (G) and non-probing phase (Np) were analysed. Individual mealybugs exhibited marked variation within species and significantly differed (p ≤ .05) between species for E1e and E1. PCR-based assessments of the retention time for CSSV in viruliferous Pl. citri, Ps. longispinus and Ps. viburni fed on a non-cacao diet showed that CSSV was still detectable after 144 hours. These unusually long durations for a pathogen currently classified as a semi-persistent virus have implications for the design of non-malvaceous barrier crops currently being considered for the protection of new cacao plantings.

Differences in phosphorus retention and release in soils amended with animal manures and sewage sludge

Excessive levels of P in agricultural soils pose a threat to local water quality. This study evaluated (i) time-dependent changes in soil P sorption (expressed as a phosphorus sorption index, PSI) and P availability (as resin P) during incubation (100 d) with poultry litter, cattle slurry, sewage sludge, or KH2PO4, added on a P-equivalent basis (100 mg P kg(-1)), and (ii) the subsequent kinetics of P release, measured by repeated extractions with a mixed cation-anion exchange resin. Soil exchangeable Ca and ammonium oxalate-extractable Fe and Al were also determined at 100 d of incubation. The small decrease in P sorption in the litter and sludge treatments (25%), compared with that in the slurry and KH2PO4 treatments (52%) between 20 and 100 d of incubation was attributed partly to the formation of new adsorption sites for P. Subsequent P release was described by a power equation: Resin P = a(extraction number)(b), where the constants a and b represent resin P obtained with a single extraction and the rate of P release per resin extraction, respectively. On average, the rate of P release decreased in the order: KH2PO4 and slurry > litter > sludge, and was inversely related to exchangeable Ca content of the incubated soils (R-2 = 0.57). The slower rates of P release in the litter and sludge treatments (P < 0.001) are attributed to the large values for exchangeable Ca (1050-2640 and 1070-2710 mg kg(-1), respectively) in these amended soils. Future research concerned with short-term declines in environmentally harmful levels of P in recently amended soils should consider the differential effects of the amendments on soil P dynamics.

Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e. g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultraperformance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats.Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0±10.4%) andheart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influencedbymicrobial activitiesormodulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Nitrogen recycling through the gut and the nitrogen economy of ruminants: An asynchronous symbiosis

The extensive development of the ruminant forestomach sets apart their N economy from that of nonruminants in a number of respects. Extensive pregastric fermentation alters the profile of protein reaching the small intestine, largely through the transformation of nitrogenous compounds into microbial protein. This process is fueled primarily by carbohydrate fermentation and includes extensive recycling of N between the body and gut lumen pools. Nitrogen recycling occurs via blood and gut lumen exchanges of urea and NH3, as well as endogenous gut and secretory N entry into the gut lumen, and the subsequent digestion and absorption of microbial and endogenous protein. Factors controlling urea transfer to the gut from blood, including the contributions of urea transporters, remain equivocal. Ammonia produced by microbial degradation of urea and dietary and endogenous AA is utilized by microbial fermentation or absorbed and primarily converted to urea. Therefore, microbial growth and carbohydrate fermentation affect the extent of NH3 absorption and urea N recycling and excretion. The extensive recycling of N to the rumen represents an evolutionary advantage of the ruminant in terms of absorbable protein supply during periods of dietary protein deficiency, or asynchronous carbohydrate and protein supply, but incurs a cost of greater N intakes, especially in terms of excess N excretion. Efforts to improve the efficiency of N utilization in ruminants by synchronizing fermentable energy and N availability have generally met with limited success with regards to production responses. In contrast, imposing asynchrony through oscillating dietary protein concentration, or infrequent supplementation, surprisingly has not negatively affected production responses unless the frequency of supplementation is less than once every 3 d. In some cases, oscillation of dietary protein concentration has improved N retention compared with animals fed an equal amount of dietary protein on a daily basis. This may reflect benefits of Orn cycle adaptations and sustained recycling of urea to the gut. The microbial symbiosis of the ruminant is inherently adaptable to asynchronous N and energy supply. Recycling of urea to the gut buffers the effect of irregular dietary N supply such that intuitive benefits of rumen synchrony in terms of the efficiency of N utilization are typically not observed in practice.

Influence of gut morphology on passive transport of freshwater bryozoans by waterfowl in Donana (southwestern Spain)

Waterbirds have been proposed as important vectors for the passive dispersal of those aquatic invertebrates and plants that lack a capacity for active dispersal between isolated water bodies. We analysed the frequency of internal transport of bryozoan propagules (statoblasts) by waterbirds in Donana, Spain, by examining their presence in the intestines and ceca of dead birds and analysing the role of different aspects of gut characteristics in explaining variation in the presence/absence and abundance of statoblasts. Of the 228 samples examined, 7.9% presented intact statoblasts of Plumatella fungosa (Pallas, 1768), Plumatella emarginata Allman, 1844, and two unidentified Plumatella species. For a given bird species, individuals with heavier gizzards and shorter ceca had a lower incidence and abundance of statoblasts in the lower gut. Grit mass and intestine length were unrelated to the presence or abundance of statoblasts. Our results suggest that waterbirds frequently transport bryozoans on a local scale, with lighter gizzards and longer ceca favouring such transport. Lighter gizzards are likely to destroy fewer propagules before they reach the lower gut. Species and individuals with longer ceca are particularly good candidates for long-distance dispersal of bryozoans, given the longer passage time of propagules that enter the ceca.