Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.

The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z' = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

Exploring the Chemical Space of Macro- and Micro-Algae Using Comparative Metabolomics.

With more than 156,000 described species, eukaryotic algae (both macro- and micro-algae) are a rich source of biological diversity, however their chemical diversity remains largely unexplored. Specialised metabolites with promising biological activities have been widely reported for seaweeds, and more recently extracts from microalgae have exhibited activity in anticancer, antimicrobial, and antioxidant screens. However, we are still missing critical information on the distinction of chemical profiles between macro- and microalgae, as well as the chemical space these metabolites cover. This study has used an untargeted comparative metabolomics approach to explore the chemical diversity of seven seaweeds and 36 microalgal strains. A total of 1390 liquid chromatography-mass spectrometry (LC-MS) features were detected, representing small organic algal metabolites, with no overlap between the seaweeds and microalgae. An in-depth analysis of four Dunaliella tertiolecta strains shows that environmental factors may play a larger role than phylogeny when classifying their metabolomic profiles.

The rest repression of the neurosecretory phenotype is negatively modulated by BHC80, a protein of the BRAF/HDAC complex.

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.

Expression of the neurosecretory process in PC12 cells is governed by REST.

The neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the dominant-negative construct induced the rescue of many vesicle/exocytosis genes but not of those of chromogranins. Small dense-core vesicles, exocytized upon stimulation, were rescued when the construct-transfected defective cells were transfected also with chromograninA or treated with trichostatinA, a blocker of histone deacetylases. Our results identify REST, working by direct and indirect mechanisms, as the factor governing the maintenance of the neurosecretory process and the properties of dense-core vesicles in PC12 cells.