bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrin-mediated Signaling Events in Human Endothelial Cells
Authors: Short Sarah M. , Talbott Gregory A. , Juliano Rudolph L.
Published: July 2013

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor α, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor α activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.
The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast
Authors: Miller Rita K. , Heller Kim K. , Frisèn Lotti , Wallack Denise L. , Loayza Diego , Gammie Alison E. , Rose Mark D.
Published: July 2013

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules inkip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that thekip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δkar9Δ double mutants were viable. Conversely,kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δdhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.
A Developmentally Regulated Kinesin-related Motor Protein fromDictyostelium discoideum
Authors: de Hostos Eugenio L. , McCaffrey Gretchen , Sucgang Richard , Pierce Daniel W. , Vale Ronald D.
Published: July 2013

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive α-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes inDictyostelium. K7, expressed in Escherichia coli, supports plus end–directed microtubule motility in vitro at a speed of 0.14 μm/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.
Mutational Effect of Fission Yeast Polα on Cell Cycle Events
Authors: Bhaumik Dipa , Wang Teresa S.-F.
Published: July 2013

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations inpolα+. Germinating spores carrying either a deletion of polα+(polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with apolαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts incdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.
Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells
Authors: Wheatley Sally P. , O’Connell Christopher B. , Wang Yu-li
Published: July 2013

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinodermembryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.
Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly
Authors: Potempa Sandra , Ridley Anne J.
Published: July 2013

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.
A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex
Authors: Nguyen Thu , Vinh Dani B.N. , Crawford Douglas K. , Davis Trisha N.
Published: July 2013

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.
Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity
Authors: Moffett John , Kratz Erica , Myers Jason , Stachowiak Ewa K. , Florkiewicz Robert Z. , Stachowiak Michal K.
Published: July 2013

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.
Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2
Authors: McConalogue Karen , Corvera Carlos U. , Gamp Patrick D. , Grady Eileen F. , Bunnett Nigel W.
Published: July 2013

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Gαq/11remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.
Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid
Authors: Lochner Janis E. , Kingma Mary , Kuhn Samuel , Meliza C. Daniel , Cutler Bryan , Scalettar Bethe A.
Published: July 2013

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.
Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation
Authors: Hendzel Michael J. , Kruhlak Michael J. , Bazett-Jones David P.
Published: July 2013

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.
Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-β Are Ligands for the Integrin αvβ1
Authors: Munger John S. , Harpel John G. , Giancotti Filippo G. , Rifkin Daniel B.
Published: July 2013

The multipotential cytokine transforming growth factor-β (TGF-β) is secreted in a latent form. Latency results from the noncovalent association of TGF-β with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-β–binding protein (LTBP) produces the most common form of latent TGF-β, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-β. LTBP and the LAP propeptides of TGF-β (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-β function in the ECM, we determined whether latent TGF-β1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits αv and β1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. αvβ1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of αvβ5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was αvβ1 dependent. These results establish αvβ1 as a LAP-β1 receptor. Interactions between latent TGF-β and αvβ1 may localize latent TGF-β to the surface of specific cells and may allow the TGF-β1 gene product to initiate signals by both TGF-β receptor and integrin pathways.
Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules inParamecium
Authors: Bré Marie-Hélène , Redeker Virginie , Vinh Joëlle , Rossier Jean , Levilliers Nicolette
Published: July 2013

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules ofParamecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with aParamecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.
Coronary microvascular dysfunction is an early feature of cardiac involvement in patients with Anderson–Fabry disease
Authors: Tomberli Benedetta , Cecchi Franco , Sciagrà Roberto , Berti Valentina , Lisi Francesca , Torricelli Francesca , Morrone Amelia , Castelli Gabriele , Yacoub Magdi H. , Olivotto Iacopo
Published: July 2013

Aims

Male patients with Anderson–Fabry disease (AFD) often exhibit cardiac involvement, characterized by LV hypertrophy (LVH), associated with severe coronary microvascular dysfunction (CMD). Whether CMD is present in patients without LVH, particularly when female, remains unresolved. The aim of the study was to investigate the presence of CMD by positron emission tomography (PET) in AFD patients of both genders, with and without evidence of LVH.

Methods and results

We assessed myocardial blood flow following dipyridamole infusion (Dip‐MBF) with 13N‐labelled ammonia by PET in 30 AFD patients (age 51 ± 13 years; 18 females) and in 24 healthy controls. LVH was defined as echocardiographic maximal LV wall thickness ≥13 mm. LVH was present in 67% of patients (n = 20; 10 males and 10 females). Dip‐MBF was reduced in all patients compared with controls (1.8 ± 0.5 and 3.2 ± 0.5 mL/min/g, respectively, P < 0.001). For both genders, flow impairment was most severe in patients with LVH (1.4 ± 0.5 mL/min/g in males and 1.9 ± 0.5 mL/min/g in females), but was also evident in those without LVH (1.8 ± 0.3 mL/min/g in males and 2.1 ± 0.4 mL/min/g in females; overall P = 0.064 vs. patients with LVH). Analysis of variance (ANOVA) for the 17 LV segments showed marked regional heterogeneity of MBF in AFD (F = 4.46, P < 0.01), with prevalent hypoperfusion of the apical region. Conversely, controls showed homogeneous LV perfusion (F = 1.25, P = 0.23).

Conclusions

Coronary microvascular function is markedly impaired in AFD patients irrespective of LVH and gender. CMD may represent the only sign of cardiac involvement in AFD patients, with potentially important implications for clinical management.
Right ventricular hypertrophy and failure abolish cardioprotection by ischaemic pre‐conditioning
Authors: Andersen Asger , Povlsen Jonas Agerlund , Bøtker Hans Erik , Nielsen‐Kudsk Jens Erik
Published: July 2013

Aims

We aimed to investigate the response to ischaemia–reperfusion (IR) and ischaemic pre‐conditioning (IPC) in the hypertrophic and failing right heart.

Methods and results

Male Wistar rats underwent sham operation, moderate pulmonary trunk banding (mPTB), or severe PTB (sPTB). Four weeks after surgery, hearts were quick‐frozen (n = 28) for biochemical analysis of key salvage pathways or isolated and perfused in a Langendorff set‐up (n = 46). We randomized perfused hearts to IPC (2 × 5 min of global ischaemia) or no preceding ischaemia (CON), before 40 min of global ischaemia and 120 min of reperfusion. The infarct size/area at risk (IS/AAR) ratio and post‐ischaemic right ventricular (RV) function were used to evaluate the effect of IPC. mPTB induced compensated RV hypertrophy and sPTB induced RV hypertrophy with failure. Hypertrophy of the right ventricle increased IS in hearts from mPTB and sPTB animals compared with sham (IS/AAR, 73.1 ± 2.9% and 59.3 ± 2.4% vs. 35.6 ± 2.9%, P < 0.0001). IPC reduced IS in sham and mPTB hearts (IS/AAR, 35.6 ± 2.9% vs. 17.4 ± 1.2% and 73.1 ± 2.9% vs. 56.9 ± 3.5%, P < 0.01) and improved recovery of RV contractile function. IPC did not alter IS/AAR (59.3 ± 2.4% and 59.3 ± 2.9%, P = 0.999) or haemodynamic recovery in sPTB hearts. RV cyclic guanosine monophosphate (cGMP) and the cGMP‐dependent protein kinase were increased after sPTB.

Conclusion

Right ventricular hypertrophy increases IR injury. Cardioprotection by IPC is abolished in the failing but not the compensated hypertrophic right ventricle of the rat heart.
Species-Specific PCR Assays for DifferentiatingHeterodera filipjeviandH. avenae
Authors: Yan Guiping , Smiley Richard W. , Okubara Patricia A. , Skantar Andrea M.
Published: July 2013

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.
Evaluating Treatments and Spray Application for the Protection of Grapevine Pruning Wounds from Infection byEutypa lata
Authors: Sosnowski Mark R. , Loschiavo Adrian P. , Wicks Trevor J. , Scott Eileen S.
Published: July 2013

Eutypa dieback threatens the sustainability of vineyards worldwide and limited treatments are available for control of the disease in grapevine. Following the loss of the effective benzimidazole fungicides, benomyl and carbendazim, there is a need to identify alternatives for the protection of pruning wounds against infection by ascospores of Eutypa lata. In this study, 24 fungicide and natural treatments were evaluated in the laboratory and field. Tebuconazole and carbendazim were the most effective fungicides for reducing colonization of pruning wounds inoculated with E. lata. Pyrimethanil and fluazinam also provided some control but were less effective than tebuconazole at the rates tested. Other treatments, such as cyprodinil + fludioxionil, pyraclostrobin, a garlic extract, and lactoferrin, reduced colonization of wounds by E. lata but require further evaluation at higher concentrations. Carbendazim applied to pruning wounds using tractor-driven sprayers reduced the incidence of pruning wound infection by E. lata to levels similar to that achieved by application with a paint brush.
Accuracy of Plateau Pressure and Stress Index to Identify Injurious Ventilation in Patients with Acute Respiratory Distress Syndrome
Authors: Terragni Pier Paolo , Filippini Claudia , Slutsky Arthur S. , Birocco Alberto , Tenaglia Tommaso , Grasso Salvatore , Stripoli Tania , Pasero Daniela , Urbino Rosario , Fanelli Vito , Faggiano Chiara , Mascia Luciana , Ranieri V. Marco
Published: July 2013

Background:

Guidelines suggest a plateau pressure (P

plat
) of 30 cm H2O or less for patients with acute respiratory distress syndrome, but ventilation may still be injurious despite adhering to this guideline. The shape of the curve plotting airway pressure versus time (S

tress

I

ndex
) may identify injurious ventilation. The authors assessed accuracy of P

plat
and S

tress

I

ndex
to identify morphological indexes of injurious ventilation.

Methods:

Indexes of lung aeration (computerized tomography) associated with injurious ventilation were used as a “reference standard.” Threshold values of P

plat
and S

tress

I

ndex
were determined assessing the receiver-operating characteristics (“training set,” N = 30). Accuracy of these values was assessed in a second group of patients (“validation set,” N = 20). P

plat
and S

tress

I

ndex
were partitioned between respiratory system (Pplat,Rs and S

tress

I

ndex
,
rs
) and lung (P

plat
,
l
and S

tress

I

ndex
,
l
; esophageal pressure; “physiological set,” N = 50).

Results:

Sensitivity and specificity of P

plat
of greater than 30 cm H2O were 0.06 (95% CI, 0.002–0.30) and 1.0 (95% CI, 0.87–1.00). P

plat
of greater than 25 cm H2O and a S

tress

I

ndex
of greater than 1.05 best identified morphological markers of injurious ventilation. Sensitivity and specificity of these values were 0.75 (95% CI, 0.35–0.97) and 0.75 (95% CI, 0.43–0.95) for P

plat
greater than 25 cm H2O versus 0.88 (95% CI, 0.47–1.00) and 0.50 (95% CI, 0.21–0.79) for S

tress

I

ndex
greater than 1.05. Pplat,Rs did not correlate with P

plat
,
l
(R
2 = 0.0099); S

tress

I

ndex
,
rs
and S

tress

I

ndex
,
l
were correlated (R
2 = 0.762).

Conclusions:

The best threshold values for discriminating morphological indexes associated with injurious ventilation were Pplat,Rs greater than 25 cm H2O and S

tress

I

ndex
,
rs
greater than 1.05. Although a substantial discrepancy between Pplat,Rs and P

plat
,
l
occurs, S

tress

I

ndex
,
rs
reflects S

tress

I

ndex
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Incidence, Paris Classification, and Follow‐up in a Nationwide Incident Cohort of Pediatric Patients With Inflammatory Bowel Disease
Authors: Müller Katalin E. , Lakatos Péter L. , Arató András , Kovács Judit B. , Várkonyi Ágnes , Szűcs Dániel , Szakos Erzsébet , Sólyom Enikő , Kovács Márta , Polgár Marianne , Nemes Éva , Guthy Ildikó , Tokodi István , Tóth Gergely , Horváth Ágnes , Tárnok András , Csoszánszki Noémi , Balogh Márta , Vass Noémi , Bódi Piroska , Dezsőfi Antal , Gárdos László , Micskey Éva , Papp Mária , Cseh Áron , Szabó Dolóresz , Vörös Péter ,   Hungarian IBD Registry Group (HUPIR), Veres Gabor
Published: July 2013

Objectives:

The aim of the study was to evaluate the incidence, baseline disease characteristics, and disease location based on the Paris classification in pediatric inflammatory bowel disease (IBD) in the Hungarian nationwide inception cohort. In addition, 1‐year follow‐up with therapy was analyzed.

Methods:

From January 1, 2007 to December 31, 2009, newly diagnosed pediatric patients with IBD were prospectively registered. Twenty‐seven pediatric gastroenterology centers participated in the data collection ensuring the data from the whole country. Newly diagnosed patients with IBD younger than 18 years were reported. Disease location was classified according to the Paris classification.

Results:

A total of 420 patients were identified. The incidence rate of pediatric IBD was 7.48/105 (95% confidence interval [CI] 6.34/105–8.83/105). The incidence for Crohn disease (CD) was 4.72/105 (95% CI 3.82–5.79), for ulcerative colitis (UC) 2.32/105 (95% CI 1.71–3.09), and for IBD‐unclassified 0.45/105 (95% CI 0.22–0.84). Most common location in CD was L3 (58.7%); typical upper gastrointestinal abnormalities (ulcer, erosion and aphthous lesion) were observed in 29.9%. Extensive colitis in patients with UC (E4, proximal to hepatic flexure) was the most common disease phenotype (57%), whereas only 5% of children had proctitis. A total of 18.6% of patients had ever severe disease (S1). Frequency of azathioprine administration at diagnosis was 29.5% in patients with CD, and this rate increased to 54.6% (130/238) at 1‐year follow‐up. In UC, only 3.3% received azathioprine initially, and this rate elevated to 22.5% (25/111). Use of corticosteroid decreased from 50% to 15.3% in patients with UC. Rate of bowel resection in patients with CD during the first year of follow‐up was 5%.

Conclusions:

The incidence of pediatric IBD in Hungary was among the higher range reported. This is the first large, nationwide incident cohort analyzed according to the Paris classification, which is a useful tool to determine the characteristic pediatric CD phenotype.
Management of Portal Hypertension in Children With Portal Vein Thrombosis
Authors: Giouleme Olga , Theocharidou Eleni
Published: July 2013

Portal vein thrombosis (PVT) is a common cause of portal hypertension in children. Predisposing conditions for PVT are obscure in more than half of the cases. Variceal bleeding and splenomegaly are the most frequent initial manifestations. Radiologic imaging studies are the mainstay for diagnosis. Treatment includes pharmacologic, endoscopic, and surgical modalities. β‐Adrenergic blockers are not routinely used in children because of unproven efficacy and significant adverse effects. Endoscopic methods, such as sclerotherapy and endoscopic variceal ligation (EVL), are highly effective in the treatment of acute variceal bleeding and eradication of varices. EVL is the treatment of choice because of minimal complications and the need for few endoscopic sessions. EVL facilitates portal decompression either by the formation of collateral vessels or by surgical portosystemic shunting, when vessels grow to the proper diameter for anastomosis. Surgical portosystemic shunts are reserved for refractory cases because of significant complications and technical difficulties. Transjugular portosystemic shunts have an emerging role in the management of portal hypertension caused by PVT. PVT may occur in the posttransplant setting, but optimal management is not defined yet.
Estimation of Fish and ω‐3 Fatty Acid Intake in Pediatric Nonalcoholic Fatty Liver Disease
Authors: St‐Jules David E. , Watters Corilee A. , Brunt Elizabeth M. , Wilkens Lynne R. , Novotny Rachel , Belt Patricia , Lavine Joel E. ,   Nonalcoholic Steatohepatitis Clinical Research Network
Published: July 2013

Aims:

Fish and ω‐3 fatty acids are reported to be beneficial in pediatric nonalcoholic fatty liver disease (NAFLD), but no studies have assessed their relation to histological severity. The objectives of this study were to evaluate the dietary intake of fish and ω‐3 fatty acids in children with biopsy‐proven NAFLD, and examine their association with serological and histological indicators of disease.

Methods:

This was a cross‐sectional analysis of 223 children (6–18 years) who participated in the Treatment of Nonalcoholic Fatty Liver Disease in Children trial or the NAFLD Database study conducted by the Nonalcoholic Steatohepatitis Clinical Research Network. The distribution of fish and ω‐3 fatty acid intake was determined from responses to the Block Brief 2000 Food Frequency Questionnaire, and analyzed for associations with serum alanine aminotransferase, histological features of fatty liver disease, and diagnosis of steatohepatitis after adjusting for demographic, anthropometric, and dietary variables.

Results:

The minority of subjects consumed the recommended 8 ounces of fish per week (22/223 [10%]) and 200 mg of long‐chain ω‐3 fatty acids per day (12/223 [5%]). Lack of fish and long‐chain ω‐3 fatty acid intake was associated with greater portal (P = 0.03 and P = 0.10, respectively) and lobular inflammation (P = 0.09 and P = 0.004, respectively) after controlling for potential confounders.

Conclusions:

Fish and ω‐3 fatty acid intake was insufficient in children with NAFLD, which may increase susceptibility to hepatic inflammation. Patients with pediatric NAFLD should be encouraged to consume the recommended amount of fish per week.
Designing artificial photosynthetic devices using hybrid organic–inorganic modules based on polyoxometalates
Authors: Symes Mark D. , Cogdell Richard J. , Cronin Leroy
Published: July 2013

Artificial photosynthesis aims at capturing solar energy and using it to produce storable fuels. However, while there is reason to be optimistic that such approaches can deliver higher energy conversion efficiencies than natural photosynthetic systems, many serious challenges remain to be addressed. Perhaps chief among these is the issue of device stability. Almost all approaches to artificial photosynthesis employ easily oxidized organic molecules as light harvesters or in catalytic centres, frequently in solution with highly oxidizing species. The ‘elephant in the room’ in this regard is that oxidation of these organic moieties is likely to occur at least as rapidly as oxidation of water, meaning that current device performance is severely curtailed. Herein, we discuss one possible solution to this problem: using self-assembling organic–polyoxometalate hybrid structures to produce compartments inside which the individual component reactions of photosynthesis can occur without such a high incidence of deleterious side reactions.
Silicon solar cells: state of the art
Authors: Green Martin A.
Published: July 2013

The vast majority of photovoltaic (PV) solar cells produced to date have been based on silicon wafers, with this dominance likely to continue well into the future. The surge in manufacturing volume over the last decade has resulted in greatly decreased costs. Multiple companies are now well below the US$1 W
−1
module manufacturing cost benchmark that was once regarded as the lowest possible with this technology. Despite these huge cost reductions, there is obvious scope for much more, as the polysilicon source material becomes more competitively priced, the new ‘quasi-mono’ and related controlled crystallization directional solidification processes are brought fully online, the sizes of ingot produced this way increase, wafer slicing switches to much quicker diamond impregnated approaches and cell conversion efficiencies increase towards the 25 per cent level. This makes the US Government's ‘SunShot’ target of US$1 W
−1
installed system cost by 2020 very achievable with silicon PVs. Paths to lower cost beyond this point are also explored.
Integrated detection of natural antisense transcripts using strand-specific RNA sequencing data
Authors: Li Song , Liberman Louisa M. , Mukherjee Neelanjan , Benfey Philip N. , Ohler Uwe
Published: July 2013

Pairs of RNA molecules transcribed from partially or entirely complementary loci are called cis-natural antisense transcripts (cis-NATs), and they play key roles in the regulation of gene expression in many organisms. A promising experimental tool for profiling sense and antisense transcription is strand-specific RNA sequencing (ssRNA-seq). To identify cis-NATs using ssRNA-seq, we developed a new computational method based on a model comparison framework that incorporates the inherent variable efficiency of generating perfectly strand-specific libraries. Applying the method to new ssRNA-seq data from whole-root and cell-type–specific Arabidopsis libraries confirmed most of the known cis-NAT pairs and identified 918 additional cis-NAT pairs. Newly identified cis-NAT pairs are supported by polyadenylation data, alternative splicing patterns, and RT-PCR validation. We found 209 cis-NAT pairs that have opposite expression levels in neighboring cell types, implying cell-type–specific roles for cis-NATs. By integrating a genome-wide epigenetic profile of Arabidopsis, we identified a unique chromatin signature of cis-NATs, suggesting a connection between cis-NAT transcription and chromatin modification in plants. An analysis of small-RNA sequencing data showed that ∼4% of cis-NAT pairs produce putative cis-NAT–induced siRNAs. Taken together, our data and analyses illustrate the potential for multifaceted regulatory roles of plant cis-NATs.
Current saturation in free-air ionization chambers with chopped synchrotron radiation
Authors: Nariyama Nobuteru
Published: July 2013

Saturation curves for free-air ionization chambers with electrode gap widths of 4.2, 8.4 and 18 mm were obtained for 10 and 15 keV undulator synchrotron radiation thinned with a 230 Hz rotating-disk chopper. Ion recombination in free-air ionization chambers was found to be inversely proportional to the applied electric field, and an expression that satisfactorily reproduced the ion-recombination rate is determined. A comparison of the expressions for continuous and pulsed X-rays revealed that chopped high-intensity X-rays require a higher voltage to attain saturation when the product of the pulse width and electric field exceeds a value that depends on the X-ray energy. This behaviour was observed explicitly for 10 keV X-rays in measurements with the ionization chamber placed before and after the chopper.
Home range and resource selection by animals constrained by linear habitat features: an example ofBlakiston's fish owl
Authors: Slaght Jonathan C. , Horne Jon S. , Surmach Sergei G. , Gutiérrez R.J.
Published: July 2013

Typically in resource selection studies, the spatial extent of a home range is defined first and then the available resources within that perimeter are estimated. However, the home ranges (or habitats) of some animals are constrained by linear environmental features (e.g. rivers, shorelines). Traditional home range estimators often overestimate home range extent for such species, which can lead to spurious estimation of resource availability and selection.

We used a synoptic model of space use to explicitly account for resource selection of a species constrained by linear features in its environment to compare with traditional home range estimators. We used the endangered Blakiston's fish owlBubo blakistoniin the Russian Far East as our example.

Mean annual home range size (± standard error) was more than three times larger when using kernel methods (30·3 ± 15·1 km2) than when using the synoptic model (9·4 ± 2·0 km2,n = 7).

Fish owls showed strong selection for areas within valleys, closer to waterways, closer to patches of permanently open water and with greater channel complexity than available sites.

Synthesis and applications. The synoptic model solves a long‐standing problem in home range and resource selection studies because it provides an objective way to estimate the space use of a species whose habitat is constrained by linear features in its environment. Improvements in the accuracy of such estimations can lead to identification of important resources across landscapes, the development of more rigorous site‐specific or landscape‐scale management plans, and to scientifically defensible conservation or threat mitigation measures.
The New National Quality Framework: Quantifying Some of the Effects on Labour Supply, Child Care Demand and Household Finances for Two‐Parent Households
Authors: Breunig Robert V. , Gong Xiaodong , Trott Declan
Published: July 2013

New regulations to improve the quality of early childhood education and care came into force in Australia in 2012. Using a simultaneous, structural model of labour supply and child care demand we predict the effects on the labour supply of partnered women, on demand for child care and on household finances for two‐parent households in Australia. Using estimated cost impacts of this new National Quality Framework from government and non‐government sources, we find modest effects on household behaviour. For a mid‐range cost scenario, we find that partnered women's labour force participation will decrease by just over one‐half of one percentage point, a change of less than 1 per cent. Working hours for partnered women decrease by 20 minutes or approximately 2 per cent. Household disposable income decreases by $12.50, a 0.6 per cent decrease. Given widespread agreement about the benefits of investing in children, these quantitatively small effects for two‐parent households strengthen the case for the National Quality Framework.