bluuebunny/crossref_metadata_2025 huggingface dataset:
The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis
Authors: Patel Ramesh , Hsu Shih-Chi , Bédard Jocelyn , Inoue Kentaro , Jarvis Paul
Published: July 2008

β-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other β-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.
Molecular and Functional Analyses Support a Role of Ornithine-δ-Aminotransferase in the Provision of Glutamate for Glutamine Biosynthesis during Pine Germination
Authors: Cañas Rafael A. , Villalobos David P. , Díaz-Moreno Sara M. , Cánovas Francisco M. , Cantón Francisco R.
Published: July 2008

We report the molecular characterization and functional analysis of a gene (PsδOAT) from Scots pine (Pinus sylvestris) encoding Orn-δ-aminotransferase (δ-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other δ-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsδOAT encodes a functional δ-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsδOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of δ-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsδOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsδOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that δ-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.
Identification of the Wax Ester Synthase/Acyl-Coenzyme A:Diacylglycerol Acyltransferase WSD1 Required for Stem Wax Ester Biosynthesis in Arabidopsis
Authors: Li Fengling , Wu Xuemin , Lam Patricia , Bird David , Zheng Huanquan , Samuels Lacey , Jetter Reinhard , Kunst Ljerka
Published: July 2008

Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C16 and C18) or very-long-chain (C20 and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.
Interactions of Two Transcriptional Repressors and Two Transcriptional Activators in Modulating Gibberellin Signaling in Aleurone Cells
Authors: Zou Xiaolu , Neuman Dawn , Shen Qingxi J.
Published: July 2008

Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b α-amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the α-amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley aleurone cells.
Geminivirus-Mediated Gene Silencing fromCotton Leaf Crumple VirusIs Enhanced by Low Temperature in Cotton
Authors: Tuttle John R. , Idris A.M. , Brown Judith K. , Haigler Candace H. , Robertson Dominique
Published: July 2008

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar ‘Deltapine 5415’ bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30°C/26°C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22°C/18°C. However, endogenous gene silencing decreased at 30°C/26°C. There was an approximately 7 d delay in the onset of gene silencing at 22°C/18°C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.
The Coenzyme A Biosynthetic Enzyme Phosphopantetheine Adenylyltransferase Plays a Crucial Role in Plant Growth, Salt/Osmotic Stress Resistance, and Seed Lipid Storage
Authors: Rubio Silvia , Whitehead Lynne , Larson Tony R. , Graham Ian A. , Rodriguez Pedro L.
Published: July 2008

Coenzyme A (CoA) is an essential cofactor in the metabolism of both prokaryotic and eukaryotic organisms and a universal five-step pathway is utilized to synthesize CoA from pantothenate. Null mutations in two of the five steps of this pathway led to embryo lethality and therefore viable reduction-of-function mutations are required to further study its role in plant biology. In this article, we have characterized a viable Arabidopsis (Arabidopsis thaliana) T-DNA mutant affected in the penultimate step of the CoA biosynthesis pathway, which is catalyzed by the enzyme phosphopantetheine adenylyltransferase (PPAT). This ppat-1 knockdown mutation showed an approximately 90% reduction in PPAT transcript levels and was severely impaired in plant growth and seed production. The sum of CoA and acetyl-CoA levels was severely reduced (60%–80%) in ppat-1 seedlings compared to wild type, and catabolism of storage lipids during seedling establishment was delayed. Conversely, PPAT overexpressing lines showed, on average, approximately 1.6-fold higher levels of CoA + acetyl-CoA levels, as well as enhanced vegetative and reproductive growth and salt/osmotic stress resistance. Interestingly, dry seeds of overexpressing lines contained between 35% to 50% more fatty acids than wild type, which suggests that CoA biosynthesis plays a crucial role in storage oil accumulation. Finally, biochemical analysis of the recombinant PPAT enzyme revealed an inhibitory effect of CoA on PPAT activity. Taken together, these results suggest that the reaction catalyzed by PPAT is a regulatory step in the CoA biosynthetic pathway that plays a key role for plant growth, stress resistance, and seed lipid storage.
Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm
Authors: Satoh Hikaru , Shibahara Kensuke , Tokunaga Takashi , Nishi Aiko , Tasaki Mikako , Hwang Seon-Kap , Okita Thomas W. , Kaneko Nanae , Fujita Naoko , Yoshida Mayumi , Hosaka Yuko , Sato Aya , Utsumi Yoshinori , Ohdan Takashi , Nakamura Yasunori
Published: July 2008

Plastidial phosphorylase (Pho1) accounts for ∼96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.
RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery
Authors: Dou Daolong , Kale Shiv D. , Wang Xia , Jiang Rays H.Y. , Bruce Nathan A. , Arredondo Felipe D. , Zhang Xuemin , Tyler Brett M.
Published: July 2008

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER–containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.
Calmodulin7 Plays an Important Role as Transcriptional Regulator inArabidopsisSeedling Development
Authors: Kushwaha Ritu , Singh Aparna , Chattopadhyay Sudip
Published: July 2008

Although calmodulin (CaM) is known to play multiple regulatory roles in eukaryotes, its direct function as transcriptional regulator is unknown. Furthermore, the physiological functions of CaM are largely unknown in plants. Here, we show that one of the four Arabidopsis thaliana CaM isoforms, CAM7, is a transcriptional regulator that directly interacts with the promoters of light-inducible genes and promotes photomorphogenesis. CAM7 overexpression causes hyperphotomorphogenic growth and an increase in the expression of light-inducible genes. Mutations in CAM7 produce no visible effects on photomorphogenic growth, indicating likely redundant gene functions. However, cam7 mutants display reduced expression of light-inducible genes, and cam7 hy5 double mutants show an enhancement of the hy5 phenotype. Moreover, overexpression of CAM7 can partly suppress the hy5 phenotype, indicating that the two factors work together to control light-induced seedling development. The mutational and transgenic studies, together with physiological analyses, illustrate the concerted function of CAM7 and HY5 basic leucine zipper transcription factor in Arabidopsis seedling development.
The 5-GHz Airport Surface Area Channel—Part I: Measurement and Modeling Results for Large Airports
Authors: Matolak David W. , Sen Indranil , Xiong Wenhui
Published: July 2008

We describe results from a channel measurement and modeling campaign for the airport surface environment in the 5-GHz band. Using a 50-MHz bandwidth test signal, thousands of power delay profiles (PDPs) were obtained and processed to develop empirical tapped-delay line statistical channel models for large airports. A log-distance path loss model was also developed. The large airport surface channel is classified into three propagation regions, and models are presented for each of the regions for two values of bandwidth. Values of the median root-mean-square (RMS) delay spread range from 500 to 1000 ns for these airports, with the 90 th percentile RMS delay spreads being approximately 1.7 ms. Corresponding correlation bandwidths (i.e., correlation value 1/2) range from approximately 1.5 MHz in non-line-of-sight (NLOS) settings to 17.5 MHz in line-of-sight (LOS) settings. Two types of statistical nonstationarity were also observed: 1) multipath component persistence and 2) propagation region transitions. We provide the multipath component probability of occurrence models and describe Markov chains that are used for modeling both phenomena. Channel tap amplitude statistics are also provided, using the flexible Weibull probability density function (pdf). This pdf was found to best fit fading tap amplitude data, particularly for frequently observed severe fading, which is characterized by fade probabilities that are worse than the commonly used Rayleigh model. Fading parameters equivalent to Nakagami-m-model values ofmnear 0.7 were often observed (withm= 1 being Rayleigh and m < 1 being worse than Rayleigh). We also provide channel tap amplitude correlation coefficients, which typically range from 0.1 to 0.4 but occasionally take values greater than 0.7.
A universal method for fishing target proteins from mixtures of biomolecules using isothermal titration calorimetry
Authors: Zhou Xingding , Sun Qingxiang , Kini R. Manjunatha , Sivaraman J.
Published: July 2008

The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell‐ or animal‐based assays, affinity labeling, solid‐phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.
The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: A molecular biological, physico‐chemical, kinetic, and vaccination study
Authors: Dewilde Sylvia , Ioanitescu A. Iulia , Kiger Laurent , Gilany Kambiz , Marden Michael C. , Van Doorslaer Sabine , Vercruysse Jozef , Pesce Alessandra , Nardini Marco , Bolognesi Martino , Moens Luc
Published: July 2008

The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single‐domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N‐termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high‐spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano‐met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal‐site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen‐binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three‐dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three‐dimensional structure of Pa.ep. determined earlier. The set of distal‐site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.
The novel p53 isoform “delta p53” is a misfolded protein and does not bind the p21 promoter site
Authors: García‐Alai Maria M. , Tidow Henning , Natan Eviatar , Townsley Fiona M. , Veprintsev Dmitry B. , Fersht Alan R.
Published: July 2008

The tumor suppressor p53 can be expressed as different isoforms because of promoter selection and mRNA editing. One isoform, “delta p53” (Δp53), results from what would be an unusual alternative splicing of exons 7/8 of the p53 gene, conserving the reading frame and generating a novel protein with proposed transcriptional activity essential for the intra S‐phase checkpoint. Here, we show that the deletion of the 66 residues that correspond to strand β10 and the C‐terminal helix of the core domain and the interconnecting linker to the tetramerization domain occurring in the Δp53 isoform leads to a misfolded and unstable protein, prone to form soluble aggregates, which does not bind the p21 promoter site. The complex of coexpressed Δp53 and flp53 is soluble in vitro and binds poorly to DNA. Our results provide a structural explanation for the dominant‐negative effect of Δp53 and its lack of transcriptional activity.
Estimating the size and dynamics of an injecting drug user population and implications for health service coverage: comparison of indirect prevalence estimation methods
Authors: Kimber Jo , Hickman Matthew , Degenhardt Louisa , Coulson Tim , Van Beek Ingrid
Published: July 2008

Aims  (i) To compare indirect estimation methods to obtain mean injecting drug use (IDU) prevalence for a confined geographic location; and (ii) to use these estimates to calculate IDU and injection coverage of a medically supervised injecting facility.

Design  Multiple indirect prevalence estimation methods.

Setting  Kings Cross, Sydney, Australia.

Participants  IDUs residing in Kings Cross area postcodes recorded in surveillance data of the Sydney Medically Supervised Injecting Centre (MSIC) between November 2001 and October 2002.

Measurements  Two closed and one open capture–recapture (CRC) models (Poisson regression, truncated Poisson and Jolly–Seber, respectively) were fitted to the observed data. Multiplier estimates were derived from opioid overdose mortality data and a cross‐sectional survey of needle and syringe programme attendees. MSIC client injection frequency and the number of needles and syringes distributed in the study area were used to estimate injection prevalence and injection coverage.

Findings  From three convergent estimates, the mean estimated size of the IDU population aged 15–54 years was 1103 (range 877–1288), yielding a population prevalence of 3.6% (2.9–4.3%). Mean IDU coverage was 70.7% (range 59.1–86.7%) and the mean adjusted injection coverage was 8.8% (range 7.3–10.8%). Approximately 11.3% of the total IDU population were estimated to be new entrants to the population per month.

Conclusions  Credible local area IDU prevalence estimates using MSIC surveillance data were obtained. MSIC appears to achieve high coverage of the local IDU population, although only an estimated one in 10 injections occurs at MSIC. Future prevalence estimation efforts should incorporate open models to capture the dynamic nature of IDU populations.
Acute liver failure following therapeutic paracetamol administration in patients with muscular dystrophies
Authors: Pearce B. , Grant I. S.
Published: July 2008

Clinically significant liver damage in patients taking therapeutic doses of paracetamol is very rare. We report two cases of fulminant hepatic failure caused by therapeutic (4 g.day−1) paracetamol administration on our Intensive Care Unit. Both patients had a muscular dystrophy and presented with a chest infection on a background of endstage neuromuscular respiratory failure. We also noted one further similar case in the literature and suggest a relationship between muscular dystrophy and paracetamol‐induced hepatotoxicity. In this report we discuss in detail possible mechanisms that may account for this apparent association, which include altered pharmacokinetics, reduced glutathione stores, malnutrition and hypoxic injury.
Patients' understanding of the reasons for starting and discontinuing inhaled corticosteroids
Authors: Menckeberg Tanja T. , Bouvy Marcel L. , Bracke Madelon , Hugtenburg Jacqueline G. , Lammers Jan‐Willem , Raaijmakers Jan A. M.
Published: July 2008

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Early discontinuation of treatment in new users of inhaled corticosteroids (ICS) has been widely discussed, but patients' reasons for this have not been investigated in depth.

WHAT THIS STUDY ADDS

• In contrast to what might have been expected, the majority of patients mentioned a wide range of symptoms or conditions as the reason for the start of ICS therapy.

• There is no clear indication for prescribing ICS in these patients.

• Most of these conditions are self limiting and may be expected to be of short duration, consequently a decrease in symptoms was the main and justifiable reason for discontinuing ICS.

• However, a non‐negligible proportion of patients has significant residual symptoms and seems to be in need of continuous ICS use.

AIM Although early discontinuation of treatment in new users of inhaled corticosteroids (ICS) has been widely discussed, the reasons for stopping have not been investigated in depth. We aimed to describe reasons for discontinuation from a patient's perspective in relation to their experience of symptoms at the time of the investigation.

METHODS A cross‐sectional study among new users that discontinued ICS use in the Netherlands was performed. Patients were interviewed by telephone, aiming to identify the symptoms for which they were prescribed ICS, the reasons for discontinuing treatment and the respiratory symptoms patients still experienced at the time of the survey. In addition, automated dispensing records of all patients were retrieved.

RESULTS From 287 eligible patients, 230 (80.1%) were interviewed. Only 22 patients (9.6%) mentioned asthma as the reason for a first ICS prescription. A decrease in symptoms was the main reason for discontinuation (45%). Thirty patients (13%) reported clinically significant residual symptoms. These patients reported more seasonal variation of symptoms and were more often prescribed short‐acting β2‐agonists.

CONCLUSIONS The majority of patients mentioned a wide range of symptoms and conditions, other than asthma or chronic obstructive pulmonary disease, as the reason for the start of ICS therapy. Most of these conditions may be expected to be of short duration. Not surprisingly a decrease in symptoms was the main, and justifiable, reason for discontinuing ICS. However, a non‐negligible proportion of patients reported residual symptoms that suggest the need of continued ICS use. Physicians and pharmacists could cooperate in identifying those patients for which ICS are really indicated and motivate them to continue the use of ICS.
Evaluation of minimal residual disease in multiple myeloma patients by fluorescent‐polymerase chain reaction: the prognostic impact of achieving molecular response
Authors: Martínez‐Sánchez Pilar , Montejano Laura , Sarasquete Maria Eugenia , García‐Sanz Ramón , Fernández‐Redondo Elena , Ayala Rosa , Montalbán María Angeles , Martínez Rafael , García Laraña José , Alegre Adrian , Hernández Belen , Lahuerta Juan José , Martínez‐López Joaquín
Published: July 2008

This study aimed to standardize a simple molecular method for evaluating the response to treatment in multiple myeloma (MM) patients after high dose chemotherapy. Fifty three patients enrolled in the GEM2000 protocol were studied for minimal residual disease (MRD) using both fluorescent‐polymerase chain reaction (F‐PCR) and flow cytometry. Most patients had achieved complete remission or very good response after autologous stem cell transplantation. The molecular analysis of immunoglobulin gene rearrangements at diagnosis and during the follow‐up was carried out by F‐PCR according to the Biomed‐2 protocols. F‐PCR could be used in 91% of the patients and the results were similar to flow cytometry. F‐PCR was able to identify a group of patients with a better prognosis [progression‐free survival (PFS) 67·86% in patients with negative F‐PCR vs. 28%; P = 0·001], even amongst patients who achieved a complete response with negative immunofixation (PFS 75% vs. 25%; P = 0·002). Multivariate analysis identified the F‐PCR result as the only variable to show a prognostic value when PFS was analysed. F‐PCR of DHJ and light chain rearrangements of immunoglobulin genes is a feasible method for evaluating MRD in MM patients after intensive therapy. Achieving molecular response by F‐PCR shows prognostic value.
Recent advances in the understanding of inherited sideroblastic anaemia
Authors: Camaschella Clara
Published: July 2008

Sideroblastic anaemia includes a heterogeneous group of rare conditions, characterized by decreased haem synthesis and mitochondrial iron overload, which are diagnosed by the presence of ringed sideroblasts in the bone marrow aspirate. The most frequent form is X‐linked sideroblastic anaemia, caused by mutations of delta‐aminolevulinic acid synthase 2 (ALAS2), the enzyme that catalyses the first and regulatory step of haem synthesis in erythroid precursors and is post‐transcriptionally controlled by the iron regulatory proteins. Impaired haem production causes variable degrees of anaemia and mitochondrial iron accumulation as ringed sideroblasts. The heterogeneity and complexity of sideroblastic anaemia is explained by an increasing number of recognized molecular defects. New forms have been recognized as being linked to the deficient function of mitochondrial proteins involved in iron–sulphur cluster biogenesis, such as ABCB7 and GLRX5, which are extremely rare but represent important biological models. Local mitochondrial iron overload is present in all sideroblastic anaemias, whereas systemic iron overload occurs only in the forms because of primary or secondary deficiency of ALAS2.
Methylene blue‐photoinactivated plasma versus quarantine fresh frozen plasma in thrombotic thrombocytopenic purpura: a multicentric, prospective cohort study
Authors: Del Río‐Garma Julio , Alvarez‐Larrán Alberto , Martínez Clara , Muncunill Josep , Castellà Dolors , De La Rubia Javier , Zamora Concepción , Corral Mercedes , Viejo Aurora , Peña Francisco , Rodríguez‐Vicente Pilar , Contreras Enric , Arbona Cristina , Ramírez Consuelo , Garcia‐Erce José A. , Alegre Adrián , Mateo José , Pereira Arturo
Published: July 2008

Plasma exchange (PE) with plasma infusion is the treatment of choice for thrombotic thrombocytopenic purpura (TTP) but doubts remain as to whether all kinds of plasma are equally effective. A multicentric cohort study was conducted to compare methylene blue‐photoinactivated plasma (MBPIP) with quarantine fresh frozen plasma (qFFP) in the treatment of TTP. One hundred and two episodes of idiopathic TTP were included; MBPIP was used in 63 and qFFP in 39. The treatment schedule consisted of daily PE and costicosteroids, and the main end‐point was remission status on day 8. Patients treated with MBPIP required more PEs (median: 11 vs. 5, P = 0·002) and a larger volume of plasma (median: 485 ml/kg vs. 216 ml/kg, P = 0·007) to achieve a remission, and presented more recrudescences while on PE therapy (29 of 63 vs. 8 of 39, P = 0·02) than those receiving qFFP. After adjustment for possible confounding factors, the use of MBPIP was associated with a lower likelihood of remission on day 8 [Odds ratio (OR): 0·17; 95% confidence interval (CI): 0·06–0·47] and a higher risk of recrudescence while on treatment (OR: 4·2; 95% CI: 1·6–10·8). In conclusion, MBPIP is less effective than qFFP in the treatment of TTP.
Migration velocity analysis for tilted transversely isotropic media
Authors: Behera Laxmidhar , Tsvankin Ilya
Published: July 2008

Tilted transversely isotropic formations cause serious imaging distortions in active tectonic areas (e.g., fold‐and‐thrust belts) and in subsalt exploration. Here, we introduce a methodology for P‐wave prestack depth imaging in tilted transversely isotropic media that properly accounts for the tilt of the symmetry axis as well as for spatial velocity variations.

For purposes of migration velocity analysis, the model is divided into blocks with constant values of the anisotropy parameters ε and δ and linearly varying symmetry‐direction velocity VP0 controlled by the vertical (kz) and lateral (kx) gradients. Since determination of tilt from P‐wave data is generally unstable, the symmetry axis is kept orthogonal to the reflectors in all trial velocity models. It is also assumed that the velocity VP0 is either known at the top of each block or remains continuous in the vertical direction. The velocity analysis algorithm estimates the velocity gradients kz and kx and the anisotropy parameters ε and δ in the layer‐stripping mode using a generalized version of the method introduced by Sarkar and Tsvankin for factorized transverse isotropy with a vertical symmetry axis.

Synthetic tests for several models typical in exploration (a syncline, uptilted shale layers near a salt dome and a bending shale layer) confirm that if the symmetry‐axis direction is fixed and VP0 is known, the parameters kz, kx, ε and δ can be resolved from reflection data. It should be emphasized that estimation of ε in tilted transversely isotropic media requires using nonhyperbolic moveout for long offsets reaching at least twice the reflector depth. We also demonstrate that application of processing algorithms designed for a vertical symmetry axis to data from tilted transversely isotropic media may lead to significant misfocusing of reflectors and errors in parameter estimation, even when the tilt is moderate (30°). The ability of our velocity analysis algorithm to separate the anisotropy parameters from the velocity gradients can be also used in lithology discrimination and geologic interpretation of seismic data in complex areas.
Velocity variations in carbonate rocks due to dual porosity and wave‐induced fluid flow
Authors: Agersborg Remy , Johansen Tor Arne , Jakobsen Morten
Published: July 2008

Stiffness variations in carbonates may be described as resulting from different concentrations of flat compliant pores or cracks, which can have a significant effect on the effective stiffness and acoustic properties (e.g., velocities and attenuations) of dry as well as saturated carbonates, although they carry extremely little porosity. As shown in this paper, the effects of dual porosity and wave‐induced fluid flow or pore pressure communication may also play a significant role. On the basis of a previously published T‐matrix approach to model the effective viscoelastic properties of cracked porous media, we illustrate the (frequency‐dependent) effects of wave‐induced fluid flow (mainly squirt flow) or pore pressure communication for a model structure consisting of a mixture of fluid‐saturated porous grains and fluid‐saturated cavities (vugs, etc.) that are embedded in a solid matrix associated with carbonates. We assume that the pores within the porous grains are decoupled from the pores in the solid matrix (and possibly saturated with different fluids) but that each pore system at the micro and/or mesoscale may or may not be connected. For each of four different connectivity models, we present numerical results for four different cases of microstructure (that emphasize the importance of cracks and flat compliant pores). Our numerical results indicate that the velocity and attenuation spectra of carbonates vary significantly, even when the crack density and all other volume concentrations are constant.
Between‐cultivar variability in breaking force of components of perennial ryegrass (Lolium perenne)
Authors: Bryant R. H. , Nicol A. M. , Wilson F. E. , Sedcole J. R.
Published: July 2008

The variation in force required to break the leaves and pseudostems of perennial ryegrass (Lolium perenne) was measured on twelve New Zealand commercial and pre‐release cultivars using a punch and die apparatus which enabled up to 100 simultaneous breaks per sample. Perennial ryegrass cultivars were sampled from 2‐year‐old pure swards in two studies carried out in spring and autumn. In the first study, the forces required to break old and young leaves, and pseudostems, of six cultivars ranging in heading date and ploidy were compared at a designated morphological stage during spring growth. Force was measured as Newtons (N) mg−1 dry matter (DM) and the difference between the force required to break leaves and pseudostems was significant (P < 0·01). The youngest emerged leaf required a greater force by 0·13 than the oldest green leaf (5·60 and 4·90 N mg−1 DM for young and old leaf respectively). The force required to break leaves (average of young and old leaves) of 5·25 N mg−1 DM was 0·12 greater than the force required to break pseudostems (4·59 N mg−1 DM). This difference was consistent across most cultivars. In the second study, the force required to break leaves of ten cultivars was tested daily over three autumn periods when all cultivars were sampled at a regrowth age of 2 weeks. The cultivar Nevis was used in each period whilst the other cultivars were used in one period. When averaged across periods, cultivars could be separated into three groups of high, medium and low forces required to break leaves. The difference between the means of the high and low groups was 0·33 with a range from 3·29 to 4·91 N mg−1 DM.
Assessing yield and yield stability of remnant populations of Cenchrus ciliaris L. in arid Tunisia: developing a blueprint for initiating native seed production
Authors: Visser M. , M’seddi K. , Chaïeb M. , Neffati M.
Published: July 2008

Native seed needs to be sourced locally yet contain ample genetic variation. A blueprint is developed for assembling and assessing native plant material at one eco‐geographical site prior to seed multiplication for restoring degraded drylands of North Africa. To satisfy the ‘local yet ample’ criterion, the feasibility of selecting a limited number of high‐yielding genotypes within one starter collection of Cenchrus ciliaris L. in Presaharian Tunisia (100–200 mm annual rainfall) was tested and compared to outcomes with similar work on its ecological complement, Stipa lagascae R. & Sch. Results showed that a limited number of high‐yielding genotypes can easily produce quality native seed because of the high genetic diversity of the starter material. Results also showed that there was a high potential to increase the rain‐use efficiency of degraded drylands using this seed. Seven ground rules are suggested when building the starter collection: (i) carefully delimit the region for sourcing seed; (ii) work with individual plants; (iii) carefully choose the collection site; (iv) replicate plant material as much as is feasible; (v) match evaluation methods to resource constraints; (vi) wait for conditions of maximum resource availability before culling; and (vii) apply a two‐tiered selection strategy.
The use of prophylaxis in 2663 children and adults with haemophilia: results of the 2006 Canadian national haemophilia prophylaxis survey
Authors: BISS T. T. , CHAN A. K. , BLANCHETTE V. S. , IWENOFU L. N. , MCLIMONT M. , CARCAO M. D. ,   FOR THE ASSOCIATION OF HEMOPHILIA CLINIC DIRECTORS OF CANADA (AHCDC) AND THE CANADIAN ASSOCIATION OF NURSES IN HEMOPHILIA CARE (CANHC)
Published: July 2008

Summary. Prophylaxis is standard of care for boys with severe haemophilia A. Indications for prophylaxis in adulthood, non‐severe haemophilia A, haemophilia B and haemophilia with inhibitors are less well defined. This survey, conducted in 2006, aimed to describe prophylaxis use in patients of all ages and severities with haemophilia A or haemophilia B in Canada. Data on 2663 individuals (2161 haemophilia A; 502 haemophilia B), including 78 inhibitor‐positive patients, were returned by 22/25 Canadian haemophilia treatment centres. This represented 98% of the Canadian haemophilia population. Frequency of prophylaxis use, defined as infusion of factor VIII/IX concentrate at least once weekly for ≥45 weeks of the year, was highest in individuals with severe haemophilia A (69%). It was lower in individuals with severe haemophilia B (32%), moderate haemophilia A (18%) or B (5%) and mild haemophilia A (1%) or B (1%). Among individuals with severe haemophilia A, the frequency of prophylaxis use was 84% in children (≤18 years) and 55% in adults (>18 years). Thirteen per cent of inhibitor‐positive individuals were receiving prophylaxis with bypassing agents. Comparison with data obtained from a 2002 Canadian survey showed a greater use of prophylaxis in children ≤5 years of age with severe haemophilia A (73% vs. 49%). Prophylaxis is no longer confined to children with severe haemophilia A, but is used in a significant proportion of adults with severe haemophilia A and individuals with severe haemophilia B or moderate haemophilia A. Prophylaxis is being started earlier in boys with severe haemophilia A.
A diagnostic dilemma: endodontic lesion or odontogenic keratocyst? A case presentation
Authors: Pace R. , Cairo F. , Giuliani V. , Prato L. P. , Pagavino G.
Published: July 2008

Aim  To present a clinical case of odontogenic keratocyst (OKC) simulating a lateral periodontal cyst.

Summary  A 39‐year‐old female complaining of swelling and pain in the left mandibular premolar area was found to have a radiolucent lesion between teeth 34 and 35 (FDI). Both teeth had incomplete root fillings, and orthograde re‐treatment of both premolars was performed. At 2‐year follow‐up, radiographic examination showed an increase in the radiolucent defect with respect to the previous examination. A surgical treatment of tooth 34 was then performed, with histological examination of the lesion. Histological features were consistent with an OKC, and the lesion was successfully treated by complete enucleation and application of Carnoy’s solution. At 2‐year follow‐up, no clinical signs or symptoms were found and the radiolucent area had disappeared.

Key learning points • Odontogenic keratocysts may mimic endodontic lesions.

• Clinicians should carefully review their cases and consider surgical intervention with biopsy in cases that do not heal.
In‐depth hardness profiles of Stainless Steel and Ni‐Ti endodontic instrument cross‐sections by nano‐indentation
Authors: Zinelis S. , Akhtar R. , Tsakiridis P. , Watts D. C. , Silikas N.
Published: July 2008

Aim To evaluate the in‐depth hardness profiles of Stainless Steel (SS) and nickel titanium (Ni‐Ti) endodontic instrument cross‐sections using a nano‐indentation technique.

Methodology Three SS (Reamer, K and Hedström) and three Ni‐Ti (ProFile, NRT and Liberator) instruments were studied. After embedding and metallographic preparation the in‐depth hardness profiles of instrument cross‐sections were measured starting from the cutting surface towards the centre to a depth of 2000 nm using an MTS XP nanoindenter with a Berkovich diamond indenter. The results of hardness measurements of outer (near to cutting edge) and inner locations were statistically analyzed by two‐wayanovafollowed by SNK test (α = 0.05).

Results For all instrument cross‐sections the maximum hardness was obtained at the outer surface followed by hardness attenuation towards the centre of the cross section. The statistical analysis of hardness classified the instruments, for both outer and innermost locations, to the following decreasing order: Reamer > K > Hedström > Profile > NRT shank (without thermal treatment) > NRT tip (with thermal treatment) > Liberator. The maximal hardness, at the outer surface of endodontic instruments, can be attributed to the residual stresses developed due to cutting and thermal effects during the manufacturing process. The increased outer layer hardness may have a beneficial effect on the cutting ability and wear resistance of endodontic instruments.

Conclusions All endodontic instruments had a decrease in hardness towards their centre. This implies that the surface hardness of contemporary endodontic instruments is significantly enhanced by the consequences of manufacturing processes.
Phosphatidylinositol‐3‐kinase inhibitor LY 294002 blocks Streptococcus mutans‐induced interleukin (IL)‐6 and IL‐8 gene expression in odontoblast‐like cells
Authors: Dommisch H. , Steglich M. , Eberhard J. , Winter J. , Jepsen S.
Published: July 2008

Aim  To investigate the involvement of the phosphatidylinositol‐3‐kinase (PI3K) in interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) gene expression in odontoblast‐like cells when exposed to heat‐killed Streptococcus mutans.

Methodology  Cultured human odontoblast‐like cells (Dulbecco’s modified Eagle’s medium) were pre‐treated with a specific inhibitor for PI3K (LY 294002) and subsequently stimulated with heat‐killed S. mutans for 6 and 24 h. After stimulation, RNA was extracted from the cells and cDNA synthesis was performed. Gene expression of IL‐6 and IL‐8 was analysed by real‐time polymerase chain reaction and normalized to the gene expression of beta‐actin. Cell survival was determined for stimulation experiments.

Results  The gene expression of IL‐6 and IL‐8 was significantly increased in response to heat‐killed S. mutans (P = 0.002 and P < 0.001, respectively). After 6 h, the mRNA expression of IL‐6 and IL‐8 was significantly higher than after 24 h of stimulation (P = 0.019 and P < 0.001, respectively). Pre‐treatment with the inhibitor LY 294002 blocked the induced gene expression of IL‐6 and IL‐8 (P = 0.002 and P < 0.001, respectively). No differences in viable cell counts were found after stimulation with heat‐killed S. mutans and/or pre‐treatment with LY 294002 compared with the unstimulated control.

Conclusion  Gene expression of IL‐6 and IL‐8 was induced by heat‐killed S. mutans via signalling pathways mediated by PI3K. These findings indicate that odontoblasts respond to cariogenic bacteria with increased gene expression of pro‐inflammatory mediators and hence participate in immune processes.