Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/PmmsA and PpHpdR/PhpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/PhpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications

High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo

Neuroimaging in anxiety disorders

Neuroimaging studies have gained increasing importance in validating neurobiological network hypotheses for anxiety disorders. Functional imaging procedures and radioligand binding studies in healthy subjects and in patients with anxiety disorders provide growing evidence of the existence of a complex anxiety network, including limbic, brainstem, temporal, and prefrontal cortical regions. Obviously, “normal anxiety” does not equal “pathological anxiety” although many phenomena are evident in healthy subjects, however to a lower extent. Differential effects of distinct brain regions and lateralization phenomena in different anxiety disorders are mentioned. An overview of neuroimaging investigations in anxiety disorders is given after a brief summary of results from healthy volunteers. Concluding implications for future research are made by the authors

Bone regeneration in surgically created defects filled with autogenous bone: an epifluorescence microscopy analysis in rats

Although the search for the ideal bone substitute has been the focus of a large number of studies, autogenous bone is still the gold standard for the filling of defects caused by pathologies and traumas, and mainly, for alveolar ridge reconstruction, allowing the titanium implants installation. OBJECTIVES: The aim of this study was to evaluate the dynamics of autogenous bone graft incorporation process to surgically created defects in rat calvaria, using epifluorescence microscopy. MATERIAL AND METHODS: Five adult male rats weighing 200-300 g were used. The animals received two 5-mm-diameter bone defects bilaterally in each parietal bone with a trephine bur under general anesthesia. Two groups of defects were formed: a control group (n=5), in which the defects were filled with blood clot, and a graft group (n=5), in which the defects were filled with autogenous bone block, removed from the contralateral defect. The fluorochromes calcein and alizarin were applied at the 7th and 30th postoperative days, respectively. The animals were killed at 35 days. RESULTS: The mineralization process was more intense in the graft group (32.09%) and occurred mainly between 7 and 30 days, the period labeled by calcein (24.66%). CONCLUSIONS: The fluorochromes showed to be appropriate to label mineralization areas. The interfacial areas between fluorochrome labels are important sources of information about the bone regeneration dynamics