Replicative intermediates of maize streak virus found during leaf development

Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombinationdependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR. © 2010 SGM.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

Comment on "Local accumulation times for source, diffusion, and degradation models in two and three dimensions" [J. Chem. Phys. 138, 104121 (2013)]

In a recent paper, Gordon, Muratov, and Shvartsman studied a partial differential equation (PDE) model describing radially symmetric diffusion and degradation in two and three dimensions. They paid particular attention to the local accumulation time (LAT), also known in the literature as the mean action time, which is a spatially dependent timescale that can be used to provide an estimate of the time required for the transient solution to effectively reach steady state. They presented exact results for three-dimensional applications and gave approximate results for the two-dimensional analogue. Here we make two generalizations of Gordon, Muratov, and Shvartsman’s work: (i) we present an exact expression for the LAT in any dimension and (ii) we present an exact expression for the variance of the distribution. The variance provides useful information regarding the spread about the mean that is not captured by the LAT. We conclude by describing further extensions of the model that were not considered by Gordon,Muratov, and Shvartsman. We have found that exact expressions for the LAT can also be derived for these important extensions...

The function and mechanisms of action of ghrelin and obestatin in ovarian cancer

In this study, we have demonstrated that the preproghrelin derived hormones, ghrelin and obestatin, may play a role in ovarian cancer. Ghrelin and obestatin stimulated an increase in cell migration in ovarian cancer cell lines and may play a role in cancer progression. Ovarian cancer is the leading cause of death among gynaecological cancers and is the sixth most common cause of cancer-related deaths in women in developed countries. As ovarian cancer is difficult to diagnose at a low tumour grade, two thirds of ovarian cancers are not diagnosed until the late stages of cancer development resulting in a poor prognosis for the patient. As a result, current treatment methods are limited and not ideal. There is an urgent need for improved diagnostic markers, as well better therapeutic approaches and adjunctive therapies for this disease. Ghrelin has a number of important physiological effects, including roles in appetite regulation and the stimulation of growth hormone release. It is also involved in regulating the immune, cardiovascular and reproductive systems and regulates sleep, memory and anxiety, and energy metabolism. Over the last decade, the ghrelin axis, (which includes the hormones ghrelin and obestatin and their receptors), has been implicated in the pathogenesis of many human diseases and it may t may also play an important role in the development of cancer. Ghrelin is a 28 amino acid peptide hormone that exists in two forms. Acyl ghrelin (usually referred to as ghrelin), has a unique n-octanoic acid post-translational modification (which is catalysed by ghrelin O-acyltransferase, GOAT), and desacyl ghrelin, which is a non-octanoylated form. Octanoylated ghrelin acts through the growth hormone secretagogue receptor type 1a (GHSR1a). GHSR1b, an alternatively spliced isoform of GHSR, is C-terminally truncated and does not bind ghrelin. Ghrelin has been implicated in the pathophysiology of a number of diseases Obestatin is a 23 amino acid, C-terminally amidated peptide which is derived from preproghrelin. Although GPR39 was originally thought to be the obestatin receptor this has been disproven, and its receptor remains unknown. Obestatin may have as diverse range of roles as ghrelin. Obestatin improves memory, inhibits thirst and anxiety, increases pancreatic juice secretion and has cardioprotective effects. Obestatin also has been shown to regulate cell proliferation, differentiation and apoptosis in some cell types. Prior to this study, little was known regarding the functions and mechanisms of action ghrelin and obestatin in ovarian cancer. In this study it was demonstrated that the full length ghrelin, GHSR1b and GOAT mRNA transcripts were expressed in all of the ovarian-derived cell lines examined (SKOV3, OV-MZ-6 and hOSE 17.1), however, these cell lines did not express GHSR1a. Ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for ghrelin, obestatin, and GOAT, but not GHSR1a, or GHSR1b. No correlations between cancer grade and the level of expression of these transcripts were observed. This study demonstrated for the first time that both ghrelin and obestatin increase cell migration in ovarian cancer cell lines. Treatment with ghrelin (for 72 hours) significantly increased cell migration in the SKOV3 and OV-MZ-6 ovarian cancer cell lines. Ghrelin (100 nM) stimulated cell migration in the SKOV3 (2.64 +/- 1.08 fold, p <0.05) and OV-MZ-6 (1.65 +/- 0.31 fold, p <0.05) ovarian cancer cell lines, but not in the representative normal cell line hOSE 17.1. This increase in migration was not accompanied by an increase in cell invasion through Matrigel. In contrast to other cancer types, ghrelin had no effect on proliferation. Ghrelin treatment (10nM) significantly decreased attachment of the SKOV3 ovarian cancer cell line to collagen IV (24.7 +/- 10.0 %, p <0.05), however, there were no changes in attachment to the other extracellular matrix molecules (ECM) tested (fibronectin, vitronectin and collagen I), and there were no changes in attachment to any of the ECM molecules in the OV-MZ-6 or hOSE 17.1 cell lines. It is, therefore, unclear if ghrelin plays a role in cell attachment in ovarian cancer. As ghrelin has previously been demonstrated to signal through the ERK1/2 pathway in cancer, we investigated ERK1/2 signalling in ovarian cancer cell lines. In the SKOV3 ovarian cancer cell line, a reduction in ERK1/2 phosphorylation (0.58 fold +/- 0.23, p <0.05) in response to 100 nM ghrelin treatment was observed, while no significant change in ERK1/2 signalling was seen in the OV-MZ-6 cell line with treatment. This suggests that this pathway is unlikely to be involved in mediating the increased migration seen in the ovarian cancer cell lines with ghrelin treatment. In this study ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for obestatin, however, no correlation between cancer grade and level of obestatin transcript expression was observed. In the ovarian-derived cell lines studied (SKOV3, OV-MZ-6 and hOSE 17.1) it was demonstrated that the full length preproghrelin mRNA transcripts were expressed in all cell lines, suggesting they have the ability to produce mature obestatin. This is the first study to demonstrate that obestatin stimulates cell migration and cell invasion. Obestatin induced a significant increase in migration in the SKOV3 ovarian cancer cell line with 10 nM (2.80 +/- 0.52 fold, p <0.05) and 100 nM treatments (3.12 +/- 0.68 fold, p <0.05) and in the OV-MZ-6 cancer cell line with 10 nM (2.04 +/- 0.10 fold, p <0.01) and 100 nM treatments (2.00 +/- 0.37 fold, p <0.05). Obestatin treatment did no affect cell migration in the hOSE 17.1normal ovarian epithelial cell line. Obestatin treatment (100 nM) also stimulated a significant increase in cell invasion in the OV-MZ-6 ovarian cancer cell line (1.45 fold +/- 0.13, p <0.05) and in the hOSE17.1 normal ovarian cell line cells (1.40 fold +/- 0.04 and 1.55 fold +/- 0.05 respectively, p <0.01) with 10 nM and 100 nM treatments. Obestatin treatment did not stimulate cell invasion in the SKOV3 ovarian cancer cell line. This lack of obestatin-stimulated invasion in the SKOV3 cell line may be a cell line specific result. In this study, obestatin did not stimulate cell proliferation in the ovarian cell lines and it has previously been shown to have no effect on cell proliferation in the BON-1 pancreatic neuroendocrine and GC rat somatotroph tumour cell lines. In contrast, obestatin has been shown to affect cell proliferation in gastric and thyroid cancer cell lines, and in some normal cell lines. Obestatin also had no effect on attachment of any of the cell lines to any of the ECM components tested (fibronectin, vitronectin, collagen I and collagen IV). The mechanism of action of obestatin was investigated further using a two dimensional-difference in gel electrophoresis (2D-DIGE) proteomic approach. After treatment with obestating (0, 10 and 100 nM), SKOV3 ovarian cancer and hOSE 17.1 normal ovarian cell lines were collected and 2D-DIGE analysis and mass spectrometry were performed to identify proteins that were differentially expressed in response to treatment. Twenty-six differentially expressed proteins were identified and analysed using Ingenuity Pathway Analysis (IPA). This linked 16 of these proteins in a network. The analysis suggested that the ERK1/2 MAPK pathway was a major mediator of obestatin action. ERK1/2 has previously been shown to be associated with obestatin-stimulated cell proliferation and with the anti-apoptotic effects of obestatin. Activation of the ERK1/2 signalling pathway by obestatin was, therefore, investigated in the SKOV3 and OV-MZ-6 ovarian cancer cell lines using anti-active antibodies and Western immunoblots. Obestatin treatment significantly decreased ERK1/2 phosphorylation at higher obestatin concentrations in both the SKOV3 (100 nM and 1000 nM) and OV-MZ-6 (1000 nM) cell lines compared to the untreated controls. Currently, very little is known about obestatin signalling in cancer. This thesis has demonstrated for the first time that the ghrelin axis may play a role in ovarian cancer migration. Ghrelin and obestatin increased cell migration in ovarian cancer cell lines, indicating that they may be a useful target for therapies that reduce ovarian cancer progression. Further studies investigating the role of the ghrelin axis using in vivo ovarian cancer metastasis models are warranted.

Three-dimensional modeling of steel portal frame buildings

The realistic strength and deflection behavior of industrial and commercial steel portal frame buildings are understood only if the effects of rigidity of end frames and profiled steel claddings are included. The conventional designs ignore these effects and are very much based on idealized two-dimensional (2D) frame behavior. Full-scale tests of a 1212 m steel portal frame building under a range of design load cases indicated that the observed deflections and bending moments in the portal frame were considerably different from those obtained from a 2D analysis of frames ignoring these effects. Three-dimensional (3D) analyses of the same building, including the effects of end frames and cladding, were carried out, and the results agreed well with full-scale test results. Results clearly indicated the need for such an analysis and for testing to study the true behavior of steel portal frame buildings. It is expected that such a 3D analysis will lead to lighter steel frames as the maximum moments and deflections are reduced.

Adhesive characteristics of low dimensional carbon nanomaterial on actin

The biosafety of carbon nanomaterial needs to be critically evaluated with both experimental and theoretical validations before extensive biomedical applications. In this letter, we present an analysis of the binding ability of two dimensional monolayer carbon nanomaterial on actin by molecular simulation to understand their adhesive characteristics on F-actin cytoskeleton. The modelling results indicate that the positively charged carbon nanomaterial has higher binding stability on actin. Compared to crystalline graphene, graphene oxide shows higher binding influence on actin when carrying positive surface charge. This theoretical investigation provides insights into the sensitivity of actin-related cellular activities on carbon nanomaterial.

Self-organized Au nanoarrays on vertical graphenes : an advanced three-dimensional sensing platform

A three-dimensional surface enhanced Raman scattering (SERS)/plasmonic sensing platform based on plasma-enabled, catalyst-free, few-layer vertical graphenes decorated with self-organized Au nanoparticle arrays is demonstrated. This platform is viable for multiple species detection and overcomes several limitations of two-dimensional sensors.

Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

Crystal structures of the potassium and rubidium salts of (3,5-dichlorophenoxy)acetic acid: Two isotypic coordination polymers

The two-dimensional coordination polymeric structures of the hydrated potassium and rubidium salts of (3,5-dichlorophenoxy)acetic acid, (3,5-D) namely, poly[mu-aqua-bis[mu3-2-(3,5-dichlorophenoxy)acetato]potassium, [K2(C8H5Cl2O3)2 (H2O)]n (I) and poly[mu-aqua-bis[mu3-2-(3,5-dichlorophenoxy)acetato]dirubidium] [Rb2(C8H5Cl2O3)2 (H2O)]n (II), respectively have been determined and are described. The two compounds are isotypic and the polymer is based on centrosymmetric dinuclear bridged complex units. The irregular six-coordination about the metal centres comprises a bridging water molecule lying on a twofold rotation axis, the phenoxy O-atom donor and and a triple bridging carboxylate O-atom of the oxoacetate side chain of the 3,5-D ligand in a bidentate chelate mode, the second carboxy O-donor, also bridging. The K-O and Rb-O bond-length ranges are 2.7238(15)--2.9459(14) and 2.832(2)--3.050(2) \%A respectively and the K...K and Rb...Rb separations in the dinuclear unit are 4.0214(7) and 4.1289(6) \%A, respectively. Within the two-dimensional layers which lie parallel to (100), the coordinated water molecule forms an O---H...O hydrogen bond to the single bridging carboxylate O atom.