Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.

Genetic responses to phosphorus deficiency

Background: Phosphorus (P) is an essential macronutrient for plants. Plants take up P as phosphate (Pi) from the soil solution. Since little Pi is available in most soils, P fertilizers are applied to crops. However, the use of P fertilizers is unsustainable and may cause pollution. Consequently, there is a need to develop more P-use-efficient (PUE) crops and precise methods to monitor crop P-status. Scope: Manipulating the expression of genes to improve the PUE of crops could reduce their P fertilizer requirement. This has stimulated research towards the identification of genes and signalling cascades involved in plant responses to P deficiency. Genes that respond to P deficiency can be grouped into 'early' genes that respond rapidly and often non-specifically to P deficiency, or 'late' genes that impact on the morphology, physiology or metabolism of plants upon Prolonged P deficiency. Summary: The use of micro-array technology has allowed researchers to catalogue the genetic responses of plants to P deficiency. Genes whose expression is altered by P deficiency include various transcription factors, which are thought to coordinate plant responses to P deficiency, and other genes involved in P acquisition and tissue P economy. Several common cis-regulatory elements have been identified in the promoters of these genes, suggesting that their expression might be coordinated. It is suggested that knowledge of the genes whose expression changes in response to P deficiency might allow the development of crops with improved PUE, and could be used in diagnostic techniques to monitor P deficiency in crops either directly using 'smart' indicator plants or indirectly through transcript profiling. The development of crops with improved PUE and the adoption of diagnostic technology could reduce production costs, minimize the use of a non-renewable resource, reduce pollution and enhance biodiversity.

A tale of two elements: P1ncP1 and RTO

The and RT0 finite element schemes are among the most promising low order elements for use in unstructured mesh marine and lake models. They are both free of spurious elevation modes, have good dispersive properties and have a relatively low computational cost. In this paper, we derive both finite element schemes in the same unified framework and discuss their respective qualities in terms of conservation, consistency, propagation factor and convergence rate. We also highlight the impact that the local variables placement can have on the model solution. The main conclusion that we can draw is that the choice between elements is highly application dependent. We suggest that the element is better suited to purely hydrodynamical applications while the RT0 element might perform better for hydrological applications that require scalar transport calculations.

Distribution and mobility of trace elements in soils and vegetation around the mining and smelting areas of Tharsis, Riotinto and Huelva, Iberian Pyrite Belt, SW Spain

Trace elements may present an environmental hazard in the vicinity of mining and smelting activities. However, the factors controlling their distribution and transfer within the soil and vegetation systems are not always well defined. Total concentrations of up to 15,195 mg center dot kg (-1) As, 6,690 mg center dot kg(-1) Cu, 24,820 mg center dot kg(-1) Pb and 9,810 mg center dot kg(-1) Zn in soils, and 62 mg center dot kg(-1) As, 1,765 mg center dot kg(-1) Cu, 280 mg center dot kg(-1) Pb and 3,460 mg center dot kg (-1) Zn in vegetation were measured. However, unusually for smelters and mines of a similar size, the elevated trace element concentrations in soils were found to be restricted to the immediate vicinity of the mines and smelters (maximum 2-3 km). Parent material, prevailing wind direction, and soil physical and chemical characteristics were found to correlate poorly with the restricted trace element distributions in soils. Hypotheses are given for this unusual distribution: (1) the contaminated soils were removed by erosion or (2) mines and smelters released large heavy particles that could not have been transported long distances. Analyses of the accumulation of trace elements in vegetation (median ratios: As 0.06, Cu 0.19, Pb 0.54 and Zn 1.07) and the percentage of total trace elements being DTPA extractable in soils (median percentages: As 0.06%, Cu 15%, Pb 7% and Zn 4%) indicated higher relative trace element mobility in soils with low total concentrations than in soils with elevated concentrations.

Factors affecting distribution and mobility of trace elements (Cu, Pb, Zn) in a perennial grapevine (Vitis vinifera L.) in the Champagne region of France

Soil and Vitis vinifera L (coarse and fine roots, leaves, berries) concentration and geochemical partitioning of Cu, Pb and Zn were determined in a contaminated calcareous Champagne plot to assess their mobility and transfer. Accumulation ratios in roots remained low (0.1-0.4 for Cu and Zn, <0.05 for Pb). Differences between elements resulted from vegetation uptake strategy and soil partitioning. Copper, significantly associated with the oxidisable fraction (27.8%), and Zn with the acid soluble fraction (33.3%), could be mobilised by rhizosphere acidification and oxidisation, unlike Pb, essentially contained in the reducible fraction (72.4%). Roots should not be considered as a whole since the more reactive fine roots showed higher accumulation ratios than coarse ones. More sensitive response of fine roots, lack of correlation between chemical extraction results and vegetation concentrations, and very limited translocation to aerial parts showed that fine root concentrations should be used when assessing bioavailability. (C) 2008 Elsevier Ltd. All rights reserved.

Scale-invariant moving finite elements for nonlinear partial differential equations in two dimensions

A scale-invariant moving finite element method is proposed for the adaptive solution of nonlinear partial differential equations. The mesh movement is based on a finite element discretisation of a scale-invariant conservation principle incorporating a monitor function, while the time discretisation of the resulting system of ordinary differential equations is carried out using a scale-invariant time-stepping which yields uniform local accuracy in time. The accuracy and reliability of the algorithm are successfully tested against exact self-similar solutions where available, and otherwise against a state-of-the-art h-refinement scheme for solutions of a two-dimensional porous medium equation problem with a moving boundary. The monitor functions used are the dependent variable and a monitor related to the surface area of the solution manifold. (c) 2005 IMACS. Published by Elsevier B.V. All rights reserved.

The infrared vibration-rotation spectrum of trans and cis nitrous acid

Infrared spectra of the trans and the cis isomers of nitrous acid, both HONO and DONO, have been observed in the gas phase using a Fourier transform interferometer with a resolution of about 0.05 cm−1 from 4000 to 500 cm−1. Rotational analyses are reported on eleven of the fundamentals and some overtones.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

Impact on CVD risk of modifying milk fat to decrease intake of SFA and increase intake of cis-MUFA

Despite the acknowledged benefits of reducing SFA intake few countries within the EU meet recognised targets. Milk and dairy products represent the single largest source of dietary SFA in most countries, yet epidemiological evidence indicates that milk has cardioprotective properties such that simply reducing consumption of dairy foods to meet SFA targets may not be a sound public health approach. The present paper explores the options for replacing some of the SFA in milk fat with cis-MUFA through alteration of the diet of the dairy cow, and the evidence that such changes can improve the indicators for CHD and CVD in general for the consumer. In addition, the outcome of such changes on risk factors for CHD and CVD at the population level is examined in the light of a modelling exercise involving data for eleven EU member states. Given the current and projected costs of health care, the results indicate that urgent consideration should be given to such a strategy.