Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

Peroxisomal Alanine: Glyoxylate Aminotransferase AGT1 Is Indispensable for Appressorium Function of the Rice Blast Pathogen, Magnaporthe oryzae

The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD+)+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD+ in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria

Dephosphorylated NSSR1 Is Induced by Androgen in Mouse Epididymis and Phosphorylated NSSR1 Is Increased during Sperm Maturation

NSSR1 (Neural salient serine/arginine rich protein 1, alternatively SRp38) is a newly identified RNA splicing factor and predominantly expressed in neural tissues. Here, by Western blot analysis and immunofluorescent staining, we showed that the expression of dephosphorylated NSSR1 increased significantly during development of the caput epididymis. In adult mice, phosphorylated NSSR1 was mainly expressed in the apical side of epithelial cells, and dephosphorylated NSSR1 in caput epididymis was upregulated in a testosterone dependent manner. In addition, subcellular immunoreactive distribution of NSSR1 varied in different regions of the epididymis. With respect to the sperm, phosphorylated NSSR1 was detected in the mid-piece of the tail as well as the acrosome. Furthermore, NSSR1 was released from the sperm head during the capacitation and acrosome reaction. These findings for the first time provide the evidence for the potential roles of NSSR1 in sperm maturation and fertilization

Menu labelling and food choice in obese adults: a feasibility study.

BACKGROUND: To date research examining the benefits of menu labelling in the UK is sparse. The aim of the present study was to examine the impact of menu labelling in a UK obese population. METHODS: Using a repeated measures design, 61 patients at a tier 3 weight management service completed four questionnaires to assess their food choice (control) and behaviour change when presented with 3 menu labelling formats (calorie content; nutrient content; and energy expenditure). RESULTS: All three forms of labelling increased participants weight control concerns compared to the control condition. There was a significant difference in content of food ordered in the three menu labelling formats compared to the control condition. The calorie condition had the largest percentage decrease in calories selected followed by energy expenditure and nutrient content. However, no difference was observed between the three conditions in the desire for menu labelling in restaurants to be introduced in the UK. CONCLUSIONS: The findings suggest that menu labelling should be enforced in the UK as it is both beneficial to promoting healthy eating and in demand. This study is the first to examine menu labelling in a UK obese population using energy expenditure equivalents to provide nutritional information

MicroRNA profiling of diverse endothelial cell types

<p>Abstract</p> <p>Background</p> <p>MicroRNAs are ~22-nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. The diversity of miRNAs in endothelial cells (ECs) and the relationship of this diversity to epithelial and hematologic cells is unknown. We investigated the baseline miRNA signature of human ECs cultured from the aorta (HAEC), coronary artery (HCEC), umbilical vein (HUVEC), pulmonary artery (HPAEC), pulmonary microvasculature (HPMVEC), dermal microvasculature (HDMVEC), and brain microvasculature (HBMVEC) to understand the diversity of miRNA expression in ECs.</p> <p>Results</p> <p>We identified 166 expressed miRNAs, of which 3 miRNAs (miR-99b, miR-20b and let-7b) differed significantly between EC types and predicted EC clustering. We confirmed the significance of these miRNAs by RT-PCR analysis and in a second data set by Sylamer analysis. We found wide diversity of miRNAs between endothelial, epithelial and hematologic cells with 99 miRNAs shared across cell types and 31 miRNAs unique to ECs. We show polycistronic miRNA chromosomal clusters have common expression levels within a given cell type.</p> <p>Conclusions</p> <p>EC miRNA expression levels are generally consistent across EC types. Three microRNAs were variable within the dataset indicating potential regulatory changes that could impact on EC phenotypic differences. MiRNA expression in endothelial, epithelial and hematologic cells differentiate these cell types. This data establishes a valuable resource characterizing the diverse miRNA signature of ECs.</p

Letters and telegrams on Andrew Inglis Clark's resignation from the Braddon Ministry, Tasmania, 1897-8

Letters and telegrams on Andrew Inglis Clark's resignation from the Braddon Ministry, 1897-8, from A.H. Aspinall, E.N.C. Braddon, Gilbert E. Butler, William Cooke, P.O. Fysh, G. D'Emden, H.T. Gould, John Gunning, Charles W. Hazell, John Henry, Frederick Lodge, J.H. Macfarlane, Andrew Miller, F.J. Prichard, Richard Ross, W.H. Smith, F. Stephens, C.H. Talbot, Alfred A. Taylor, H. Thomas, G.J. Walford and J.N. Woolnough. C4/C390 (1-22

Rational steering of insulin binding specificity by intra-chain chemical crosslinking

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu I -catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormones B-chain C-terminus for its IR-B specificity

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta