A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.

Infant sensitivity to negative maternal emotional shifts: Effects of infant sex, maternal postnatal depression, and interactive style

This study examined the effects of infant sex, maternal postnatal depression, and maternal interactive style on infant sensitivity to maternal negative emotional shifts. Face-to-face interactions of 68 mother–infant dyads were analyzed at 8 and 18 weeks. Twenty-five (28%) mothers had postnatal depression. Interactions were analyzed in terms of overall maternal interactive style: “sensitive,” “anxious,” “intrusive,” and “sad.” Episodes of negative shifts in maternal emotional expression were recorded, along with expressions of infant sensitivity to these changes. Daughters of depressed mothers showed higher rates of sensitivity to maternal negative emotion whereas their sons showed lower rates, in comparison to both girl and boy infants of well mothers. While maternal interactive style had no effect on 8-week infant sensitivity to maternal negative emotional shifts, high rates of 18-week infant sensitivity were predicted by both an 8-week and a concurrent, “sad” maternal interactive style. The findings are discussed in relation to theories of emotional and interpersonal development.

Enhanced sensitivity to rapamycin following long-term oestrogen deprivation in MCF-7, T-47-D and ZR-75-1 human breast cancer cells

Human breast cancer cells (MCF-7, T-47-D and ZR-75-1) can adapt to circumvent any reduced growth rate during long-term oestrogen deprivation, and this provides three model systems to investigate mechanisms of endocrine resistance in breast cancer. In this paper we report consistent differences in the effects of three growth inhibitors following long-term oestrogen deprivation in all three cell models. Long-term oestrogen deprivation of MCF-7, T-47-D and ZR-75-1 cells resulted in reduced growth inhibition by PD98059 (2–10 µg/ml), implying a loss of dependence on mitogen-activated protein kinase pathways for growth. The growth inhibitor LY294002 (2–10 µM) inhibited growth of both oestrogen-maintained and oestrogen-deprived cells with similar dose–responses, implying continued similar dependence on phosphoinositide 3-kinase (PI3K) pathways with no alteration after adaptation to oestrogen independent growth. However, by contrast, long-term oestrogen deprivation resulted in an increased sensitivity to growth inhibition by rapamycin, which was not reduced by readdition of oestradiol. The enhanced inhibition of long-term oestrogen-deprived MCF-7-ED, T-47-D-ED and ZR-75-1-ED cell growth by combining rapamycin with LY294002 at concentrations where each alone had little effect, offers preclinical support to the development of therapeutic combinations of rapamycin analogues with other PI3K inhibitors in endocrine-resistant breast cancer.

Sensitivity analysis of a cyanobacterial growth and movement model under two different flow regimes

Bloom-forming and toxin-producing cyanobacteria remain a persistent nuisance across the world. Modelling cyanobacterial behaviour in freshwaters is an important tool for understanding their population dynamics and predicting the location and timing of the bloom events in lakes, reservoirs and rivers. A new deterministic–mathematical model was developed, which simulates the growth and movement of cyanobacterial blooms in river systems. The model focuses on the mathematical description of the bloom formation, vertical migration and lateral transport of colonies within river environments by taking into account the major factors that affect the cyanobacterial bloom formation in rivers including light, nutrients and temperature. A parameter sensitivity analysis using a one-at-a-time approach was carried out. There were two objectives of the sensitivity analysis presented in this paper: to identify the key parameters controlling the growth and movement patterns of cyanobacteria and to provide a means for model validation. The result of the analysis suggested that maximum growth rate and day length period were the most significant parameters in determining the population growth and colony depth, respectively.

Acute effects of meal fatty acid composition on insulin sensitivity in healthy post-menopausal women

Postprandial plasma insulin concentrations after a single high-fat meal may be modified by the presence of specific fatty acids although the effects of sequential meal ingestion are unknown. The aim of the present study was to examine the effects of altering the fatty acid composition in a single mixed fat-carbohydrate meal on glucose metabolism and insulin sensitivity of a second meal eaten 5 h later. Insulin sensitivity was assessed using a minimal model approach. Ten healthy post-menopausal women underwent four two-meal studies in random order. A high-fat breakfast (40 g fat) where the fatty acid composition was predominantly saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), long-chain n-3 PUFA or monounsaturated fatty acids (MUFA) was followed 5 h later by a low-fat, high-carbohydrate lunch (5.7 g fat), which was identical in all four studies. The plasma insulin response was significantly higher following the SFA meal than the other meals after both breakfast and lunch (P<0.006) although there was no effect of breakfast fatty acid composition on plasma glucose concentrations. Postprandial insulin sensitivity (SI(Oral)) was assessed for 180 min after each meal. SI(Oral) was significantly lower after lunch than after breakfast for all four test meals (P=0.019) following the same rank order (SFA < n-6 PUFA < n-3 PUFA < MUFA) for each meal. The present study demonstrates that a single meal rich in SFA reduces postprandial insulin sensitivity with 'carry-over' effects for the next meal.

A sensitivity study for water availability in the Northern Caucasus based on climate projections

A strong climatic warming is currently observed in the Caucasus mountains, which has profound impact on runoff generation in the glaciated Glavny (Main) Range and on water availability in the whole region. To assess future changes in the hydrological cycle, the output of a general circulation model was downscaled statistically. For the 21st century, a further warming by 4–7 °C and a slight precipitation increase is predicted. Measured and simulated meteorological variables were used as input into a runoff model to transfer climate signals into a hydrological response under both present and future climate forcings. Runoff scenarios for the mid and the end of the 21st century were generated for different steps of deglaciation. The results show a satisfactory model performance for periods with observed runoff. Future water availability strongly depends on the velocity of glacier retreat. In a first phase, a surplus of water will increase flood risk in hot years and after continuing glacier reduction, annual runoff will again approximate current values. However, the seasonal distribution of streamflow will change towards runoff increase in spring and lower flows in summer.

Variation in the sensitivity of DOC release between different organic soils following H2SO4 and sea-salt additions

Long-term monitoring data from eastern North America and Europe indicate a link between increased dissolved organic carbon (DOC) concentrations in surface waters over the last two decades and decreased atmospheric pollutant and marine sulphur (S) deposition. The hypothesis is that decreased acidity and ionic strength associated with declining S deposition has increased the solubility of DOC. However, the sign and magnitude of DOC trends have varied between sites, and in some cases at sites where S deposition has declined, no significant increase in DOC has been observed, creating uncertainty about the causal mechanisms driving the observed trends. In this paper, we demonstrate chemical regulation of DOC release from organic soils in batch experiments caused by changes in acidity and conductivity (measured as a proxy for ionic strength) associated with controlled SO42− additions. DOC release from the top 10 cm of the O-horizon of organo-mineral soils and peats decreased by 21–60% in response to additions of 0–437 µeq SO42− l−1 sulphuric acid (H2SO4) and neutral sea-salt solutions (containing Na+, Mg2+, Cl−, SO42−) over a 20-hour extraction period. A significant decrease in the proportion of the acid-sensitive coloured aromatic humic acids (measured by specific ultra-violet absorbance (SUVA) at 254 nm) was also found with increasing acidity (P < 0.05) in most, but not all, soils, confirming that DOC quality, as well as quantity, changed with SO42− additions. DOC release appeared to be more sensitive to increased acidity than to increased conductivity. By comparing the change in DOC release with bulk soil properties, we found that DOC release from the O-horizon of organo-mineral soils and semi-confined peats, which contained greater exchangeable aluminium (Al) and had lower base saturation (BS), were more sensitive to SO42− additions than DOC release from blanket peats with low concentrations of exchangeable Al and greater BS. Therefore, variation in soil type and acid/base status between sites may partly explain the difference in the magnitude of DOC changes seen at different sites where declines in S deposition have been similar.

Forecast impact of targeted observations: sensitivity to observation error and proximity to steep orography

For a targeted observations case, the dependence of the size of the forecast impact on the targeted dropsonde observation error in the data assimilation is assessed. The targeted observations were made in the lee of Greenland; the dependence of the impact on the proximity of the observations to the Greenland coast is also investigated. Experiments were conducted using the Met Office Unified Model (MetUM), over a limited-area domain at 24-km grid spacing, with a four-dimensional variational data assimilation (4D-Var) scheme. Reducing the operational dropsonde observation errors by one-half increases the maximum forecast improvement from 5% to 7%–10%, measured in terms of total energy. However, the largest impact is seen by replacing two dropsondes on the Greenland coast with two farther from the steep orography; this increases the maximum forecast improvement from 5% to 18% for an 18-h forecast (using operational observation errors). Forecast degradation caused by two dropsonde observations on the Greenland coast is shown to arise from spreading of data by the background errors up the steep slope of Greenland. Removing boundary layer data from these dropsondes reduces the forecast degradation, but it is only a partial solution to this problem. Although only from one case study, these results suggest that observations positioned within a correlation length scale of steep orography may degrade the forecast through the anomalous upslope spreading of analysis increments along terrain-following model levels.

Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type

BACKGROUND: Bruchid beetles, Callosobruchus species, are serious pests of economically important grain legumes; their activity in stores is often controlled by use of synthetic insecticides. Esterases are known to be involved in insecticide resistance in insects. However, there is dearth of information on esterase activity in the genus Callosobruchus. In this study we investigated the effect of species, geographical strain and food type on the variation of acetylcholinesterase (AChE) activity and its inhibition by malaoxon (malathion metabolite) using an in vitro spectrophotometric method. RESULT: AChE activity varied significantly among species and strains and also among legume type used for rearing them. Generally irrespective of species, strain or food type, the higher the AChE activity of a population, the higher its inhibition by malaoxon. C. chinensis had the highest AChE activity of the species studied and in the presence of malaoxon it had the lowest remaining AChE activity, while C. rhodesianus retained the highest activity. CONCLUSION: A firsthand knowledge of AChE activity in regional Callosobruchus in line with the prevailing food types should be of utmost importance to grain legume breeders, researchers on plant materials for bruchid control and pesticide manufacturer/applicators for a robust integrated management of these bruchids.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.