Compartmentalization of Mammalian Pantothenate Kinases

The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.     

Diaryl Disulfides as Novel Stabilizers of Tumor Suppressor Pdcd4

The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70^S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2–4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted.     

The Genealogy of Civilization

Building on insights from Freud, Nietzsche, and Norbert Elias, the author addresses the question of the construction of guilt in Judeo-Christian civilization. The author begins with a consideration of biological versus cultural reproduction, as these are figured in the Torah and in the Christian Bible, and in tha moves on to some social-structural, historical, and psychodynamic considerations of how and why civilization manages to sustain itself.     

A non-destructive assay for GUS in the media of plant tissue cultures

β-glucuronidase (GUS) can be assayed in the spent media of plant tissues transformed with some GUS gene fusions (Jefferson, 1988). This approach is based on the presence of GUS in the media of transformed plant tissues expressing the gene and can be used to monitor the progress of transformation without destruction of the tissue under study.     

Expression and Characterization of Recombinant,Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).     

Analysis of microbiota in cultures of the green microalga Tetraselmis suecica

ABSTRACT

Bacteria associated with microalgae strongly affect algal biomass and derived product yield and quality. Nevertheless, only a few studies have addressed the detailed phylogenetic characterization of bacterial communities associated with microalgae. In this study, the phycospheric bacterial communities associated with different Tetraselmis suecica F&M-M33 cultures, a green marine microalga with several industrial applications, were analysed using a metagenomic approach. The T. suecica F&M-M33 cultures used originated from the same ancestral microalgal non-axenic culture but were physically and geographically separated for years and maintained under different growing conditions. Despite the different history of the cultures, a ‘core’ bacterial community was identified, accounting for 70% of the total bacterial community and formed by at least 13 families. Among the ‘core’ operational taxonomic units (OTUs), 24 different genera were identified. Nevertheless, there was a high variability in the relative proportions of the taxa forming the ‘core’ community, indicating that the growing conditions and/or external contamination influence the relative abundance of these microorganisms. Our study allowed the identification of persistent taxa that may be used to deepen the knowledge of the complex relationship between T. suecica and its associated bacteria.     

Spatial versus Day-To-Day Within-Lake Variability in Tropical Floodplain Lake CH4 Emissions – Developing Optimized Approaches to Representative Flux Measurements

Inland waters (lakes, rivers and reservoirs) are now understood to contribute large amounts of methane (CH₄) to the atmosphere. However, fluxes are poorly constrained and there is a need for improved knowledge on spatiotemporal variability and on ways of optimizing sampling efforts to yield representative emission estimates for different types of aquatic ecosystems. Low-latitude floodplain lakes and wetlands are among the most high-emitting environments, and here we provide a detailed investigation of spatial and day-to-day variability in a shallow floodplain lake in the Pantanal in Brazil over a five-day period. CH₄ flux was dominated by frequent and ubiquitous ebullition. A strong but predictable spatial variability (decreasing flux with increasing distance to the shore or to littoral vegetation) was found, and this pattern can be addressed by sampling along transects from the shore to the center. Although no distinct day-to-day variability were found, a significant increase in flux was identified from measurement day 1 to measurement day 5, which was likely attributable to a simultaneous increase in temperature. Our study demonstrates that representative emission assessments requires consideration of spatial variability, but also that spatial variability patterns are predictable for lakes of this type and may therefore be addressed through limited sampling efforts if designed properly (e.g., fewer chambers may be used if organized along transects). Such optimized assessments of spatial variability are beneficial by allowing more of the available sampling resources to focus on assessing temporal variability, thereby improving overall flux assessments.