Characterization of the bga1-encoded glycoside hydrolase family 35 beta-galactosidase of Hypocrea jecorina with galacto-beta-D-galactanase activity

The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.     

First description of the environmental niche of the epibenthic dinoflagellate species Coolia palmyrensis,C. malayensis,and C. tropicalis (Dinophyceae) from Eastern Australia

Environmental variables such as temperature, salinity, and irradiance are significant drivers of microalgal growth and distribution. Therefore, understanding how these variables influence fitness of potentially toxic microalgal species is particularly important. In this study, strains of the potentially harmful epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis were isolated from coastal shallow water habitats on the east coast of Australia and identified using the D1‐D3 region of the large subunit (LSU) ribosomal DNA (rDNA). To determine the environmental niche of each taxon, growth was measured across a gradient of temperature (15–30°C), salinity (20–38), and irradiance (10–200 μmol photons · m^?2 · s^?1). Specific growth rates of Coolia tropicalis were highest under warm temperatures (27°C), low salinities (ca. 23), and intermediate irradiance levels (150 μmol photons · m^?2 · s^?1), while C. malayensis showed the highest growth at moderate temperatures (24°C) and irradiance levels (150 μmol photons · m^?2 · s^?1) and growth rates were consistent across the range of salinity levels tested (20–38). Coolia palmyrensis had the highest growth rate of all species tested and favored moderate temperatures (24°C), oceanic salinity (35), and high irradiance (>200 μmol photons · m^?2 · s^?1). This is the first study to characterize the environmental niche of species from the benthic harmful algal bloom genus Coolia and provides important information to help define species distributions and inform risk management.     

Rational design and characterization of bioplastics from Hermetia illucens prepupae proteins

In this study proteins extracted from prepupae of Hermetia illucens, also known as black soldier fly, are investigated as promising base for a new type of bioplastics for agricultural purposes. Design of experiments techniques are employed to perform a rational study on the effects of different combination of glycerol as plasticizer, citric acid as cross-linking agent and distilled water as solvent on the capability of proteins to form a free-standing film through casting technique, keeping as fixed the quantity of proteins. Glycerol shows interesting properties as plasticizer contributing to the formation of homogenous and free-standing film. Moreover, mechanical and thermal characterizations are performed to estimate the effect of increasing amounts of proteins on the final properties and thickness of the specimens. Proteins derived from H. illucens can be successfully employed as base for bioplastics to be employed for agricultural purposes.