Barley transcriptome analyses upon interaction with different aphid species identify thionins contributing to resistance.

Aphids are phloem-feeding insects that cause yield loss on a wide range of crops, including cereals such as barley. Whilst most aphid species are limited to one or few host species, some are able to reproduce on many plants belonging to different families. Interestingly, aphid probing behaviour can be observed on both host and non-host species, indicating that interactions take place at the molecular level that may impact host range. Here, we aimed to gain insight into the interaction of barley with aphid species differing in their ability to infest this crop by analysing transcriptional responses. Firstly, we determined colonization efficiency, settlement and probing behaviour for the aphid species Rhopalosiphum padi, Myzus persicae and Myzus cerasi, which defined host, poor-host and non-host interactions, respectively. Analyses of barley transcriptional responses revealed gene sets differentially regulated upon the different barley-aphid interactions and showed that the poor-host interaction with M. persicae resulted in the strongest regulation of genes. Interestingly, we identified several thionin genes strongly up-regulated upon interaction with M. persicae, and to a lesser extent upon R. padi interaction. Ectopic expression of two of these genes in Nicotiana benthamiana reduced host susceptibility to M. persicae, indicating that thionins contribute to defences against aphids.

Towards an understanding of the control of 'crumbly' fruit in red raspberry.

The genetic disorder known as 'crumbly' fruit is becoming a serious problem in the European raspberry industry. The study set out to examine the crumbly phenotype in a red raspberry mapping population under two environments (field and polytunnel) across six seasons in an effort to understand variability of the syndrome and to examine whether genetic factors were important and if so, whether QTL associated with the phenotype could be identified. This highlighted that seasonal, environmental (field or polytunnel) and genetic factors all influence the condition. Two QTL that are important for the genetic control of the condition have been located on linkage groups one and three, and an association with ripening time has been identified.

Including ethical considerations in models for first-trimester screening for pre-eclampsia.

Recent efforts to develop reliable and efficient early pregnancy screening programmes for pre-eclampsia have focused on combining clinical, biochemical and biophysical markers. The same model has been used for first-trimester screening for fetal aneuploidies i.e. prenatal diagnosis (PD), which is routinely offered to all pregnant women in many developed countries. Some studies suggest combining PD and pre-eclampsia screening, so women can be offered testing for a number of conditions at the same clinical visit. A combination of these tests may be practical in terms of saving time and resources; however, the combination raises ethical issues. First-trimester PD and pre-eclampsia screening entail qualitative differences which alter the requirements for disclosure, non-directedness and consent with regard to the informed consent process. This article explores the differences related to the ethical issues raised by PD and pre-eclampsia in order to elucidate which factors are relevant to deciding the type of information and consent required in each context from the perspective of the ethical principles of beneficence and autonomy. Furthermore, it argues that ensuring respect for patient autonomy is context dependent and, consequently, pre-eclampsia screening and PD should be performed independently of one another.

Increased risk of preterm delivery and pre-eclampsia in women with polycystic ovary syndrome and hyperandrogenaemia.

OBJECTIVE: To study the risk of adverse pregnancy outcomes in women with polycystic ovary syndrome (PCOS), and to examine the role of hyperandrogenaemia. DESIGN: Cohort study. SETTING: Singleton pregnancies in women with PCOS identified at a private fertility clinic during 1997-2010 and a background population including all singleton deliveries at Hvidovre Hospital, Denmark, in 2005. POPULATION: A cohort of 459 women with PCOS and a background population of 5409 women. METHODS: Obstetric outcomes were extracted from national Danish registries and odds ratios (ORs) were calculated by multiple logistic regression analysis, adjusting for age, parity, and body mass index. MAIN OUTCOME MEASURES: Risk of pre-eclampsia, preterm delivery, and small for gestational age offspring in the entire PCOS population and in a subsample with hyperandrogenaemia. RESULTS: Women with PCOS had an increased risk of preterm delivery <37 weeks of gestation (OR 2.28; 95% confidence interval, 95% CI, 1.51-3.45; P < 0.0001). The elevated risk was confined to hyperandrogenic women with PCOS: preterm delivery before 37 weeks of gestation (OR 2.78; 95% CI 1.62-4.77; P < 0.0001), and was not seen in normoandrogenic women with PCOS (OR 1.35; 95% CI 0.54-3.39; P = 0.52). The overall risk of pre-eclampsia was not elevated (OR 1.69; 95% CI 0.99-2.88; P = 0.05) compared with the background population, but was significantly increased in the hyperandrogenic subsample (OR 2.41; 95% CI 1.26-4.58; P < 0.001). The risk of small for gestational age offspring was similar in all groups. CONCLUSION: Women with PCOS had an increased risk of preterm delivery compared with the background population. The increased risk was confined to hyperandrogenic women with PCOS who had a two-fold increased risk of preterm delivery and pre-eclampsia.

Cytochrome P450 polymorphism and postoperative cognitive dysfunction.

BACKGROUND: The etiology of postoperative cognitive dysfunction (POCD) remains unclear but toxicity of anesthetic drugs and their metabolites could be important. We aimed to assess the possible association between POCD after propofol anesthesia and various phenotypes owing to polymorphisms in cytochrome P450 encoding genes. METHODS: We included patients who underwent non-cardiac surgery under total intravenous anesthesia with propofol. POCD was identified using a neuropsychological test-battery administered preoperatively, one week, and three months after surgery. Genotyping of CYP2C19*2, *3, CYP2D6*3, *4, *5 and *6 was performed using pyrosequencing, and patients were characterized according to their phenotype as ultra, extensive, intermediate, or poor metabolizers. RESULTS: In total, 337 patients with a median age of 67 years were included. 30 (9.4%) out of the 319 patients who underwent neuropsychological testing at one week had POCD, and 24 out of 307 (7.8%) had POCD at three months. None of the examined CYP2C19, 2D6 alleles, or various phenotypes were significantly associated with POCD. CONCLUSION: Polymorphisms in CYP2C19, or 2D6 genes do not seem to be related to the occurrence of cognitive dysfunction after non-cardiac surgery in patients anesthetised with propofol.

High-efficiency multiplex capillary electrophoresis single strand conformation polymorphism (multi-CE-SSCP) mutation screening of SCN5A: a rapid genetic approach to cardiac arrhythmia.

Mutations in the SCN5A gene coding for the alpha-subunit of the cardiac Na(+) ion channel cause long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, sick sinus node syndrome, progressive conduction disease, dilated cardiomyopathy and atrial standstill. These diseases exhibit variable expressivity, and identification of gene carriers is clinically important, particularly in sudden infant and adult death syndromes. The SCN5A gene comprises 28 exons distributed over 100 kbp of genomic sequence at chromosome 3p21. Disease-causing mutations are private and scattered over the DNA sequence, making it difficult to screen for specific mutations. We developed a multiplex capillary-electrophoresis single-strand conformation polymorphism (Multi-CE-SSCP) mutation screening protocol on the ABI 3100 platform and applied it to 10 previously slab-gel SSCP identified mutations and SNPs and used it to identify one novel deletion. The method is highly efficient, with a turnover of 23 patients per 24 h and a false positive rate of 0.5% of the analyzed amplicons. Each variant has a particular elution pattern, and all 20 carriers of the H558R polymorphism out of 57 persons were correctly identified. We suggest that the method could become part of routine work-up of patients with suspicious syncope and of members of families with sudden unexplained death.

Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.).

The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Exon skipping induced by cold stress in a potato invertase gene transcript.

We show that two invertase genes in potato, like most other plant invertase genes, include a very short second exon of 9 bp which encodes the central three amino acids of a motif highly conserved in invertases of diverse origin. This mini-exon is one of the smallest known in plants and pre-mRNA from these genes may be susceptible to alternative splicing, because of a potential requirement for specialized interaction with the splicing machinery to ensure correct processing for the production of a mature mRNA. No evidence of aberrant post-transcriptional processing was observed during normal invertase gene expression in potato. The fidelity of post-transcriptional processing of the pre-mRNA from one of the genes was perturbed by cold stress, resulting in the deletion of the mini-exon from some transcripts. This alternative splicing event occurred under cold stress in both leaf and stem, but was not induced by wounding. This adds an example of exon skipping and the induction of alternative processing by cold stress to the small number of transcripts which have been shown to exhibit alternative splicing in plants. The differential sensitivity of post-transcriptional processing to cold stress observed for the two transcripts examined will permit further dissection of the nucleotide sequence requirements for their accurate splicing.

Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity.

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosum L.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.

Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression.

A full-length cDNA clone encoding a potato invertase (Inv) has been isolated. It is highly related (77% nucleotide identity) to a previously characterised potato cDNA clone encoding a putative extracellular Inv. These Inv genes encode a subfamily of apoplastic enzymes which are shown to be distinct, on the basis of sequence similarity, from the related subfamily of vacuolar enzymes. In order to differentiate between the expression of the two potato genes encoding apoplastic Inv, a single-stranded conformational polymorphism (SSCP) assay was developed for products generated by reverse transcription-polymerase chain reaction (RT-PCR) utilising primers designed to amplify both potato sequences. Using this approach, we have shown that these two identified Inv from potato are expressed in a tissue-specific and developmentally regulated manner.

cDNA cloning and expression of a potato (Solanum tuberosum) invertase.

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.

Automated quality assurance and patient dosimetry in diagnostic radiology.

It has been generally considered that improved methods of quality assurance would reduce the population dose from diagnostic radiology. This paper describes the development of a computerized method of automatically monitoring tube and generator parameters to perform on-line quality assurance, whilst undertaking various patient dosimetry measurements and calculations for each exposure. The method involves interfacing a microcomputer to a microprocessor controlled X-ray generator. Details of the various interfacing methods and modifications to the X-ray unit are given. The instrument enables quality assurance to be performed for every exposure by comparing tube and generator parameters against nominal settings. The software automatically warns the operator of any deviations from accepted limiting values. When a patient is examined, details of the examination and projection are entered into a database. The exposure area product and field size are monitored for each exposure. This data, together with information on tube potential and examination/projection is used to deduce patient entrance skin dose and energy imparted. Doses to individual organs are estimated using normalized organ dose data and a knowledge of tube potential and field size.

Developmental regulation of insulin like growth factor II gene expression in the pig.

The expression of the insulin like growth factor II gene has been examined in the developing porcine embryo. It was found that IGF II transcripts were present in abundant quantities in first trimester embryos as well as in the extraembryonic tissues amnion and allantochorion. The expression of the IGF II gene was high in the fetal liver where a prominent 2.3 kB transcript and a less abundant 5.4 kB transcript were produced. However, neither of these transcripts could be traced in the adult liver. Instead we found two different IGF II transcripts with the sizes of 4.7 and 1.2 kB in the adult liver. These findings indicate that the IGF II gene is under developmental control with the possible existence of different promoters.

An instrument for monitoring the force applied by breast compression devices on mammography x-ray sets.

Automatic breast compression devices are commonly fitted to mammographic x-ray sets. The force exerted by these compression devices should be limited and periodically checked. This article describes the design and construction of an instrument for the routine assessment of the force applied by breast compression devices on mammography x-ray sets. Results obtained on four different mammography x-ray units are presented. In addition, data on the effect of compression plate design on compression force is given for one unit. The instrument has proved to be a simple and quick method of monitoring the force applied by the compression plates on mammographic x-ray units.