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.

Identification of domestication-related loci associated with flowering time and seed size in soybean with the RAD-seq genotyping method

Flowering time and seed size are traits related to domestication. However, identification of domestication-related loci/genes of controlling the traits in soybean is rarely reported. In this study, we identified a total of 48 domestication-related loci based on RAD-seq genotyping of a natural population comprising 286 accessions. Among these, four on chromosome 12 and additional two on chromosomes 11 and 15 were associated with flowering time, and four on chromosomes 11 and 16 were associated with seed size. Of the five genes associated with flowering time and the three genes associated with seed size, three genes Glyma11g18720, Glyma11g15480 and Glyma15g35080 were homologous to Arabidopsis genes, additional five genes were found for the first time to be associated with these two traits. Glyma11g18720 and Glyma05g28130 were co-expressed with five genes homologous to flowering time genes in Arabidopsis, and Glyma11g15480 was co-expressed with 24 genes homologous to seed development genes in Arabidopsis. This study indicates that integration of population divergence analysis, genome-wide association study and expression analysis is an efficient approach to identify candidate domestication-related genes.

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.

Inheritance of time to flowering in cowpea (Vigna unguiculata (L.) Walp.)

Time to flowering and maturity is an important adaptive feature in annual crops, including cowpeas (Vigna unguiculata (L.) Walp.). In West and Central Africa, photoperiod is the most important environmental variable affecting time to flowering in cowpea. The inheritance of time from sowing to flowering (f) in cowpeas was studied by crossing a photoperiod-sensitive genotype Kanannnado to a photoperiod-insensitive variety IT97D-941-1. Sufficient seed of F-1, F-2, F-3 and backcross populations were generated. The parental, F-1, F-2, F-3 and the backcross populations were screened for f under long natural days (mean daylength 13.4 h per day) in the field and the parents, F-1, F-2 and backcross populations under short day (10 h per day) conditions. The result of the screening showed that photoperiod in the field was long enough to delay flowering of photoperiod-sensitive genotypes. Photoperiod-sensitivity was found to be partially dominant to insensitivity. Frequency distribution of the trait in the various populations indicated quantitative inheritance. Additive (d) and additive x dominance (j) interactions were the most important gene actions conditioning time to flowering. A narrow sense heritability of 86% was estimated for this trait. This will result in 26 days gain in time to flowering with 5% selection intensity from the F-2 to F-3 generation. At least seven major gene pairs, with an average delay of 6 days each, were estimated to control time to flowering in this cross.

Heterotic and seed rate effects on nitrogen efficiencies in wheat

Four field experiments over 2 years investigated whether wheat hybrids had higher nitrogen-use efficiency (NUE) than their parents over a range of seed rates and different N regimes. There was little heterosis for total N in the above-ground biomass (NYt), but there was high-parent heterosis for grain N yields (NYg) in two of the hybrids, Hyno Esta and Hyno Rista, associated with greater nitrogen harvest index (NHI). Overall, the hybrids did not significantly increase the total dry matter produced per unit N in the above-ground crop (NUtE(t)), but did increase the grain dry matter per unit N in the above ground crop (NUtE(g)). The improvement in NUtE(g) was at the partial detriment of grain N concentration. Heterosis for grain NYg in Hyno Esta was lower at zero-N, suggesting that it did not achieve higher yields through more efficient capture or utilization of N. The greater NHI in Hyno Esta appeared to be facilitated by both greater N uptake, and remobilization of N from vegetative tissues, after anthesis. The response of N efficiency and uptake to seed rate was dependent on N supply and season. Where N fertilizer was applied, N uptake over time was slower at the lower seed rates, but where N was withheld N capture at the lowest seed rate soon approached the N capture of the higher seed rates. During grain filling, the rate of accumulation of N into the grain increased with seed rate and the duration of N accumulation decreased with seed rate. With N applied, N yields increased to all asymptote with seed rate, when N was withheld there was little response of N yields to seed rate. In 2002, N utilization efficiency (NUtE(t) and NUtE(g)) also increased asymptotically with seed rate, but in 2003 seed rate had little effect on N utilization efficiency. When nitrogen fertilizer had not been applied, NHI consistently decreased with increasing seed rate. The timing of N application made little difference to NUE, NY, or NUtE.

Seed development, maturation and storage behaviour of Mimusops elengi L

Mass maturity (end of the seed-filling phase) occurred at about 72 days after flowering (DAF) in developing seeds of Mimusops elengi, at which time seed moisture content had declined to about 55%. The onset of ability to germinate was detected at 56 DAF and seeds showed 98% germination by 84 DAF. Tolerance of desiccation to 10% moisture content was first detected at 70 DAF and was maximal by 84 DAF. Delaying collection by a further 14 days to 98 DAF, when fruits began to be shed, reduced seed viability, particularly for seeds first dried to 10% moisture content. Hence the best time for seed collection appears to be about 14 days before fruits shed. In a separate investigation with six different seed lots, desiccation below about 8-12% moisture content reduced viability (considerably in some lots). The viability of dry seeds (below about 10% moisture content) stored hermetically was reduced at cool temperatures (5 degrees C and below), and none survived storage at sub-zero temperatures. The results suggest that Mimusops elengi shows intermediate seed storage behaviour and that the optimal hermetic seed storage environment is about 10% moisture content at 10 degrees C, while short-term, moist, aerated storage at high (40%) moisture content is also feasible.