Novel insights into ascorbate retention and degradation during the washing and post-harvest storage of spinach and other salad leaves.

Post-harvest treatments of pre-packaged salad leaves potentially cause l-ascorbate loss, but the mechanisms of ascorbate degradation remain incompletely understood, especially in planta. We explored the extent and pathways of ascorbate loss in variously washed and stored salad leaves. Ascorbate was assayed by 2,6-dichlorophenolindophenol titration, and pathways were monitored by 14C-radiolabelling followed by high-voltage electrophoresis. All leaves tested showed ascorbate loss during storage: lettuce showed the greatest percentage loss, wild rocket the least. Spinach leaves were particularly prone to losing ascorbate during washing, especially with simultaneous mechanical agitation; however, washing in the presence of hypochlorite did not significantly increase ascorbate loss. In spinach, [14C]oxalate was the major product of [14C]ascorbate degradation, suggesting that commercial washing causes oxidative stress. This study highlights that ascorbate/dehydroascorbic acid are lost via the oxidative pathway during washing and post-harvest storage of salad leaves. Thus changes to washing procedures could potentially increase the post-harvest retention of ascorbate.

Characterization of the watercress (Nasturtium officinale R. Br.; Brassicaceae) transcriptome using RNASeq and identification of candidate genes for important phytonutrient traits linked to human health.

BACKGROUND: Consuming watercress is thought to provide health benefits as a consequence of its phytonutrient composition. However, for watercress there are currently limited genetic resources underpinning breeding efforts for either yield or phytonutritional traits. In this paper, we use RNASeq data from twelve watercress accessions to characterize the transcriptome, perform candidate gene mining and conduct differential expression analysis for two key phytonutritional traits: antioxidant (AO) capacity and glucosinolate (GLS) content. RESULTS: The watercress transcriptome was assembled to 80,800 transcripts (48,732 unigenes); 71 % of which were annotated based on orthology to Arabidopsis. Differential expression analysis comparing watercress accessions with 'high' and 'low' AO and GLS resulted in 145 and 94 differentially expressed loci for AO capacity and GLS respectively. Differentially expressed loci between high and low AO watercress were significantly enriched for genes involved in plant defence and response to stimuli, in line with the observation that AO are involved in plant stress-response. Differential expression between the high and low GLS watercress identified links to GLS regulation and also novel transcripts warranting further investigation. Additionally, we successfully identified watercress orthologs for Arabidopsis phenylpropanoid, GLS and shikimate biosynthesis pathway genes, and compiled a catalogue of polymorphic markers for future applications. CONCLUSIONS: Our work describes the first transcriptome of watercress and establishes the foundation for further molecular study by providing valuable resources, including sequence data, annotated transcripts, candidate genes and markers.

Diversity in global gene expression and morphology across a watercress (Nasturtium officinale R. Br.) germplasm collection: first steps to breeding.

Watercress (Nasturtium officinale R. Br.) is a nutrient intense, leafy crop that is consumed raw or in soups across the globe, but for which, currently no genomic resources or breeding programme exists. Promising morphological, biochemical and functional genomic variation was identified for the first time in a newly established watercress germplasm collection, consisting of 48 watercress accessions sourced from contrasting global locations. Stem length, stem diameter and anti-oxidant (AO) potential varied across the accessions. This variation was used to identify three extreme contrasting accessions for further analysis. Variation in global gene expression was investigated using an Affymetrix Arabidopsis ATH1 microarray gene chip, using the commercial control (C), an accession selected for dwarf phenotype with a high AO potential (dwarfAO, called 'Boldrewood') and one with high AO potential alone. A set of transcripts significantly differentially expressed between these three accessions, were identified, including transcripts involved in the regulation of growth and development and those involved in secondary metabolism. In particular, when differential gene expression was compared between C and dwarfAO, the dwarfAO was characterised by increased expression of genes encoding glucosinolates, which are known precursors of phenethyl isothiocyanate, linked to the anti-carcinogenic effects well-documented in watercress. This study provides the first analysis of natural variation across the watercress genome and has identified important underpinning information for future breeding for enhanced anti-carcinogenic properties and morphology traits in this nutrient-intense crop.

Automatic detection of regions in spinach canopies responding to soil moisture deficit using combined visible and thermal imagery.

Thermal imaging has been used in the past for remote detection of regions of canopy showing symptoms of stress, including water deficit stress. Stress indices derived from thermal images have been used as an indicator of canopy water status, but these depend on the choice of reference surfaces and environmental conditions and can be confounded by variations in complex canopy structure. Therefore, in this work, instead of using stress indices, information from thermal and visible light imagery was combined along with machine learning techniques to identify regions of canopy showing a response to soil water deficit. Thermal and visible light images of a spinach canopy with different levels of soil moisture were captured. Statistical measurements from these images were extracted and used to classify between canopies growing in well-watered soil or under soil moisture deficit using Support Vector Machines (SVM) and Gaussian Processes Classifier (GPC) and a combination of both the classifiers. The classification results show a high correlation with soil moisture. We demonstrate that regions of a spinach crop responding to soil water deficit can be identified by using machine learning techniques with a high accuracy of 97%. This method could, in principle, be applied to any crop at a range of scales.

Antioxidant assays - consistent findings from FRAP and ORAC reveal a negative impact of organic cultivation on antioxidant potential in spinach but not watercress or rocket leaves.

Watercress (Rorippa nasturtium-aquaticum), wild rocket (Diplotaxis tenuifolia), and spinach (Spinacia oleracea) are commercial crops reported to have high concentrations of antioxidants, possibly contributing to disease prevention following human consumption. Following analysis of supermarket-purchased salad leaves, we report the antioxidant content potential of these species using two comparable techniques assessing the consistency between the assays - by the ferric reducing antioxidant power (FRAP) assay and the oxygen radical absorbance capacity (ORAC) assay. The leaves were harvested from both conventionally and organically managed crops, to investigate whether organic agriculture results in improved crop quality. Watercress had the highest FRAP and ability to scavenge free radicals, followed by spinach and rocket. For watercress and rocket, there was no significant effect of organic agriculture on FRAP and ORAC, but for spinach, the antioxidant potential was reduced and this was significant at the 5% level of probability for FRAP but not ORAC, although the trend was clear in both tests. We conclude that there is variation in salad crop antioxidant potential and that FRAP and ORAC are useful techniques for measuring antioxidants in these salad crops with similar ranking for each salad crop studied.

Modification of cell wall properties in lettuce improves shelf life.

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Inversion-recovery-prepared SSFP for cardiac-phase-resolved delayed-enhancement MRI.

Delayed-enhancement magnetic resonance imaging (DE-MRI) can be used to visualize myocardial infarction (MI). DE-MRI is conventionally acquired with an inversion-recovery gradient-echo (IR-GRE) pulse sequence that yields a single bright-blood image. IR-GRE imaging requires an accurate estimate of the inversion time (TI) to null the signal from the myocardium, and a separate cine acquisition is required to visualize myocardial wall motion. Simulations were performed to examine the effects of a steady-state free precession (SSFP) readout after an inversion pulse in the setting of DE-MRI. Using these simulations, a segmented IR-SSFP sequence was optimized for infarct visualization. This sequence yields both viability and wall motion images over the cardiac cycle in a single breath-hold. Viability images at multiple effective TIs are produced, providing a range of image contrasts. In a study of 11 patients, IR-SSFP yielded infarct sizes and left ventricular ejection fractions (LVEFs) similar to those obtained by IR-GRE and standard SSFP, respectively. IR-SSFP images yielded improved visualization of the infarct-blood border because of the simultaneous nulling of healthy myocardium and blood. T(1) (*) recovery curves were extracted from IR-SSFP images and showed excellent qualitative agreement with theoretical simulations.

QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits.

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative trait loci (QTLs) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity, and breakstrength, suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life and leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index, and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer post-harvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTLs relevant to leaf development and shelf life were assessed. In total, 107 QTLs, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTLs were common in both environments. Several areas where many QTLs co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTLs for leaf biophysical properties (breakstrength, plasticity, and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTLs for leaf development, strength, and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker-assisted breeding.

MRI relaxation fluctuations in acute reperfused hemorrhagic infarction.

MRI evaluations of intramyocardial hemorrhage in acute infarction have relied on T(2) and T(2)(*) shortening only. We propose a more comprehensive evaluation of hemorrhagic infarction based on the concept that fluctuations in T(2) and T(1) relaxation in acute reperfused infarction will reflect transient edema and hemoglobin oxidative denaturation to uncompartmentalized methemoglobin. Anteroapical infarction was created via percutaneous balloon in young swine (22-25 kg, N = 12). T(2), T(1), diastolic wall thickness (DWT), and the Gd-DTPA partition coefficient (lambda) were measured on days 0, 2, and 7. DWT was elevated at 1 hr postreperfusion (128% +/- 53%, P = 0.0001), and alleviated on days 2 and 7 (48% +/- 10%, P = 0.008; 53% +/- 24%, P = 0.003). T(2) and T(1) elevations were coincident with early edema (DeltaT(2) = 55% +/- 24%, P < 0.0001; DeltaT(1) = 27% +/- 18%, P < 0.04). T(2) and T(1) were nearly normal on day 2 (DeltaT(2) = 8% +/- 8%, P = 0.27; DeltaT(1) = 0% +/- 1%, P = 0.65). On day 7, T(2) increased while T(1) decreased (DeltaT(2) = 27% +/- 16%, P = 0.005; DeltaT(1) = -14% +/- 10%, P = 0.02). Lambda was elevated by >150% at all time points (P < or = 0.002). Histology verified hemorrhagic injury. T(1) and T(2) fluctuations are consistent with transient edema, as well as hemoglobin oxidative denaturation to decompartmentalized methemoglobin. This methodological development may broaden our understanding of hemorrhagic microvascular injury and improve its detection in clinical populations.

T2 fluctuations in ischemic and post-ischemic viable porcine myocardium in vivo.

T2 relaxation can augment delayed-enhancement viability imaging because it is sensitive to tissue edema and microcirculatory oxygen state. We demonstrate the T2 'signatures' of sub-lethal ischemia and stunning in porcine myocardium perfused by the distal left anterior descending artery, by imaging during percutaneous balloon occlusion for 25 minutes and subsequent reperfusion (n = 9). Muscle displayed ischemic dysfunction and partial post-ischemic functional recovery (p < or = 0.0004), concommitant with an elevated post-ischemic T2 (deltaT2 = 27 +/- 18%, p = 0.005). TTC staining verified muscle viability. The T2 fluctuations may reflect hyperemia and tissue cellular edema in accord with the known pathophysiology of ischemic and post-ischemic yet viable muscle.

Impact of the National Service Framework for coronary heart disease on treatment and outcome of patients with acute coronary syndromes.

OBJECTIVES: To evaluate the impact the National Service Framework (NSF) for coronary heart disease has had on emergency treatment and outcomes in patients presenting with acute coronary syndromes. DESIGN: Retrospective cohort study. SETTING: Coronary care units of two district general hospitals. RESULTS: Data from 3371 patients were recorded, 1993 patients in the 27 months before the introduction of the NSF and 1378 patients in the 24 months afterwards. After the introduction of the NSF in-hospital mortality was significantly reduced (95 patients (4.8%) v 43 (3.2%), p = 0.02). This was associated with a reduction in the development of Q wave myocardial infarction (40.6% v 33.3%, p < 0.0001) and in the incidence of left ventricular failure (15.9% v 12.3%, p = 0.003). The proportion of patients receiving thrombolysis increased (69.4% v 84.7%, p < 0.0001) with a decrease in the time taken to receive it (proportion thrombolysed within 20 minutes 12.1% v 26.6%, p < 0.0001). The prescription of beta blockers (51.9% v 65.8%, p < 0.0001), angiotensin converting enzyme inhibitors (37% v 66.4%, p < 0.0001), and statins (55.2% v 72.7%, p < 0.0001) improved and the proportion of patients referred for invasive investigation increased (18.3% v 27.0%, p < 0.0001). Trend analysis showed that improvements in mortality and thrombolysis were directly associated with publication of the NSF, whereas the improvements seen in prescription of beta blockers and statins were the continuation of pre-existing trends. CONCLUSIONS: In the two years that followed publication of the NSF the initial treatment and outcome of patients presenting with acute coronary syndromes improved. Some of the improvements can be attributed to the NSF but others are continuations of pre-existing trends.

Stomatal conductance and not stomatal density determines the long-term reduction in leaf transpiration of poplar in elevated CO2.

Using a free-air CO2 enrichment (FACE) experiment, poplar trees (Populus x euramericana clone I214) were exposed to either ambient or elevated [CO2] from planting, for a 5-year period during canopy development, closure, coppice and re-growth. In each year, measurements were taken of stomatal density (SD, number mm(-2)) and stomatal index (SI, the proportion of epidermal cells forming stomata). In year 5, measurements were also taken of leaf stomatal conductance (gs, micromol m(-2) s(-1)), photosynthetic CO2 fixation (A, mmol m(-2) s(-1)), instantaneous water-use efficiency (A/E) and the ratio of intercellular to atmospheric CO2 (Ci:Ca). Elevated [CO2] caused reductions in SI in the first year, and in SD in the first 2 years, when the canopy was largely open. In following years, when the canopy had closed, elevated [CO2] had no detectable effects on stomatal numbers or index. In contrast, even after 5 years of exposure to elevated [CO2], gs was reduced, A/E was stimulated, and Ci:Ca was reduced relative to ambient [CO2]. These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest that stomatal numbers (SD and SI) had no role in determining the improved instantaneous leaf-level efficiency of water use under elevated [CO2]. We propose that altered cuticular development during canopy closure may partially explain the changing response of stomata to elevated [CO2], although the mechanism for this remains obscure.

Risedronate prevents bone loss in early postmenopausal women: a prospective randomized, placebo-controlled trial.

OBJECTIVES: To assess the efficacy and tolerability of risedronate, a pyridinyl bisphosphonate, in preventing loss of bone mineral density (BMD) of the lumbar spine and proximal femur in early postmenopausal women. METHODS: A total of 383 patients were randomly assigned to receive risedronate 2.5 or 5 mg or placebo once daily for 24 months. All patients received 1 g elemental calcium daily. BMD was measured by dual X-ray absorptiometry at baseline and at 3, 6, 12, 18, and 24 months. RESULTS: Risedronate 5 mg significantly increased BMD at the lumbar spine and femoral neck and trochanter in early postmenopausal women. Significant results were observed as early as 3 months. In the control calcium-supplemented group, BMD decreased steadily at each site throughout the study. The mean percentage change from baseline in BMD in the risedronate 5 mg group was significantly different from that in the control group at each determination at each site. At 24 months, the differences were 4.5 +/- 0.45% at the lumbar spine, 3.3 +/- 0.49% at the femoral neck, and 4.3 +/- 0.67% at the femoral trochanter. Risedronate 2.5 mg maintained BMD at each site, although the effect was less pronounced than that of risedronate 5 mg. Risedronate was well tolerated and was not associated with an increased incidence of overall or upper gastrointestinal adverse events. CONCLUSIONS: Risedronate 5 mg prevents bone loss in early postmenopausal women, is well tolerated, and represents an effective choice to maintain bone mass and prevent osteoporosis.

192IrCl acid skin burn: case report and review of the literature.

A worker was contaminated following a chemical explosion that splashed an HNO3 radioactive solution containing approximately 180 MBq (5 mCi) 192Ir onto the left side of his face. Initial efforts reduced the contamination at least fivefold. Removal of a patch of contaminated hair was necessary. Most of the contamination was fixed to the skin; only a small amount of contamination was absorbed.

NK cells from human MHC class I (HLA-B) transgenic mice do not mediate hybrid resistance killing against parental nontransgenic cells.

We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.

An Alternative Framework for Defining Mediation.

The present article provides an alternative framework for evaluating mediated relationships. From this perspective. a mediated process is a chain reaction, beginning with an independent variable that affects a mediator that in turn affects an outcome. The definition of mediation offered here, presented for stage sequences, states three conditions for establishing mediation: (a) the independent variable affects the probability of the sequence no mediator to mediator to outcome; (b) the independent variable affects the probability of a transition into the mediator stage; (c) the mediator affects the probability of a transition into the outcome stage at every level of the independent variable. This definition of mediation is compared and contrasted with the well-known definition of mediation for continuous variables discussed in Baron and Kenny (1986), Judd and Kenny (1981), and Kenny, Kashy, and Bolger (1997). The definition presented in this article emphasizes the intraindividual, time-ordered nature of mediation.

Dynamics of shear-induced redistribution of F-actin in endothelial cells in vivo.

The steady-state responses of endothelial cell F-actin distribution to changes in in vivo shear stress have been well documented. The purpose of the current work was to define the dynamics of redistribution of F-actin in the period immediately after experimental changes in shear. We used abdominal aortic coarctation in rabbits to experimentally increase shear stress downstream from the coarctation by approximately twofold. In situ staining was employed to track subsequent F-actin redistribution. Within 12-15 hours, the number of stress fibers in the central regions of the cells decreased, and some separation of junctional actin in adjacent cells occurred. Long, central stress fibers of variable thickness were evident at 24 hours, but the band of actin normally seen at the periphery of the cells could no longer be distinguished. The redistribution of F-actin was completed over the next 24 hours by an increase in thickness of central stress fibers. Restoration of normal F-actin distribution after coarctations were removed proceeded more slowly. The long, thick stress fibers that were induced by high shear were replaced by thinner or shorter microfilament bundles 48 hours after the coarctations were removed. At 72 hours, central stress fibers were primarily long, thin structures. Peripheral F-actin was not fully restored at this time. Peripheral F-actin was restored at 1 week after removal of the coarctation, but there were still more and longer stress fibers at this time than were observed in control aortas.(ABSTRACT TRUNCATED AT 250 WORDS)