A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrioanguillarum strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-29-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5- HmdC, 5-formyl-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5- HmdU), and the (59S) diastereomer of 8,59-cyclo-29-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tetfamily dioxygenase enzymes in Arabidopsis

Stabilization of long-looped i-motif DNA by polypyridyl ruthenium complexes

A spectroscopic study of the interactions of Λ- and Δ-[Ru(phen)2(dppz)]2+ with i-motif DNA containing thymine loops of various lengths. In the presence of i-motifs, the luminescence of the Λ enantiomer was enhanced much more than the Δ. Despite this, the effect of each enantiomer on i-motif thermal stability was comparable. The sequences most affected by [Ru(phen)2(dppz)]2+ were those with long thymine loops; this suggests that long-looped i-motifs are attractive targets for potential transition metal complex drugs and should be explored further in drug design

Tuber shape and eye depth variation in a diploid family of Andean potatoes.

BACKGROUND: Tuber appearance is highly variable in the Andean cultivated potato germplasm. The diploid backcross mapping population ‘DMDD’ derived from the recently sequenced genome ‘DM’ represents a sample of the allelic variation for tuber shape and eye depth present in the Andean landraces. Here we evaluate the utility of morphological descriptors for tuber shape for identification of genetic loci responsible for the shape and eye depth variation. RESULTS: Subjective morphological descriptors and objective tuber length and width measurements were used for assessment of variation in tuber shape and eye depth. Phenotypic data obtained from three trials and male–female based genetic maps were used for quantitative trait locus (QTL) identification. Seven morphological tuber shapes were identified within the population. A continuous distribution of phenotypes was found using the ratio of tuber length to tuber width and a QTL was identified in the paternal map on chromosome 10. Using toPt-437059, the marker at the peak of this QTL, the seven tuber shapes were classified into two groups: cylindrical and non-cylindrical. In the first group, shapes classified as ‘compressed’, ‘round’, ‘oblong’, and ‘long-oblong’ mainly carried a marker allele originating from the male parent. The tubers in this group had deeper eyes, for which a strong QTL was found at the same location on chromosome 10 of the paternal map. The non-cylindrical tubers classified as ‘obovoid’, ‘elliptic’, and ‘elongated’ were in the second group, mostly lacking the marker allele originating from the male parent. The main QTL for shape and eye depth were located in the same genomic region as the previously mapped dominant genes for round tuber shape and eye depth. A number of candidate genes underlying the significant QTL markers for tuber shape and eye depth were identified. CONCLUSIONS: Utilization of a molecular marker at the shape and eye depth QTL enabled the reclassification of the variation in general tuber shape to two main groups. Quantitative measurement of the length and width at different parts of the tuber is recommended to accompany the morphological descriptor classification to correctly capture the shape variation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12863-015-0213-0) contains supplementary material, which is available to authorized users

The anti-bacterial iron-restriction defence mechanisms of egg white; the potential role of three lipocalin-like proteins in resistance against Salmonella

Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identified as the most common vehicles of infection. Possibly, the major antibacterial property of egg white is iron restriction, which results from the presence of the iron-binding protein, ovotransferrin. To circumvent iron restriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelate iron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in egg white that could enhance egg-white iron restriction through sequestration of certain siderophores, including enterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterial growth via its siderophore-binding capacity in vitro. However, it remains unclear whether ex-FABP performs such a function in egg white or during bird infection. Regarding the two other lipocalins of egg white (Cal-γ and α-1-glycoprotein), there is currently no evidence to indicate that they sequester siderophores

Stressed out symbiotes:hypotheses for the influence of abiotic stress on arbuscular mycorrhizal fungi

Abiotic stress is a widespread threat to both plant and soil communities. Arbuscular mycorrhizal (AM) fungi can alleviate effects of abiotic stress by improving host plant stress tolerance, but the direct effects of abiotic stress on AM fungi are less well understood. We propose two hypotheses predicting how AM fungi will respond to abiotic stress. The stress exclusion hypothesis predicts that AM fungal abundance and diversity will decrease with persistent abiotic stress. The mycorrhizal stress adaptation hypothesis predicts that AM fungi will evolve in response to abiotic stress to maintain their fitness. We conclude that abiotic stress can have effects on AM fungi independent of the effects on the host plant. AM fungal communities will change in composition in response to abiotic stress, which may mean the loss of important individual species. This could alter feedbacks to the plant community and beyond. AM fungi will adapt to abiotic stress independent of their host plant. The adaptation of AM fungi to abiotic stress should allow the maintenance of the plant-AM fungal mutualism in the face of changing climates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00442-016-3673-7) contains supplementary material, which is available to authorized users

Non-Invasive Measurement of Hemoglobin: Assessment of Two Different Point-of-Care Technologies

Measurement of blood hemoglobin (Hb) concentration is a routine procedure. Using a non-invasive point-of-care device reduces pain and discomfort for the patient and allows time saving in patient care. The aims of the present study were to assess the concordance of Hb levels obtained non-invasively with the Pronto-7 monitor (version 2.1.9, Masimo Corporation, Irvine, USA) or with the NBM-200MP monitor (Orsense, Nes Ziona, Israel) and the values obtained from the usual colorimetric method using blood samples and to determine the source of discordance.We conducted two consecutive prospective open trials enrolling patients presenting in the emergency department of a university hospital. The first was designed to assess Pronto-7™ and the second NBM-200MP™. In each study, the main outcome measure was the agreement between both methods. Independent factors associated with the bias were determined using multiple linear regression. Three hundred patients were prospectively enrolled in each study. For Pronto-7™, the absolute mean difference was 0.56 g.L(-1) (95% confidence interval [CI] 0.41 to 0.69) with an upper agreement limit at 2.94 g.L(-1) (95% CI [2.70;3.19]), a lower agreement limit at -1.84 g.L(-1) (95% CI [-2.08;-1.58]) and an intra-class correlation coefficient at 0.80 (95% CI [0.74;0.84]). The corresponding values for the NBM-200MP™ were 0.21 [0.02;0.39], 3.42 [3.10;3.74], -3.01 [-3.32;-2.69] and 0.69 [0.62;0.75]. Multivariate analysis showed that age and laboratory values of hemoglobin were independently associated with the bias when using Pronto-7™, while perfusion index and laboratory value of hemoglobin were independently associated with the bias when using NBM-200MP™.Despite a relatively limited bias in both cases, the large limits of agreement found in both cases render the clinical usefulness of such devices debatable. For both devices, the bias is independently and inversely associated with the true value of hemoglobin.ClinicalTrials.gov NCT01321580 and NCT01321593

The Arabidopsis thaliana Brassinosteroid Receptor (AtBRI1) Contains a Domain that Functions as a Guanylyl Cyclase In Vitro

BACKGROUND: Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. PRINCIPLE FINDINGS: Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. CONCLUSIONS: Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs