Resolving cellular systems by ultra-sensitive and economical single-cell transcriptome filtering.

Single-cell transcriptomics suffer from sensitivity limits that restrict low abundance transcript identification, affects clustering and can hamper downstream analyses. Here, we describe Constellation sequencing (Constellation-Seq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximizing read utility while reducing the data sparsity and sequencing costs. The technique reliably measures changes in gene expression and was demonstrated by resolving rare dendritic cell populations from a peripheral blood mononuclear cell sample sample and exploring their biology with extreme resolution. The simple and powerful method is fully compatible with standard scRNA-Seq library preparation protocols and can be used for hypothesis testing, marker validation or investigating pathways.

Small molecule antagonism of oxysterol-induced Epstein-Barr virus induced gene 2 (EBI2) activation.

The Epstein-Barr virus induced gene 2 (EBI2) was recently identified as the first oxysterol-activated 7TM receptor. EBI2 is essential for B cell trafficking within lymphoid tissues and thus the humoral immune response in general. Here we characterize the antagonism of the non-peptide molecule GSK682753A, which blocks oxysterol-induced G-protein activation, ß-arrestin recruitment and B-cell chemotaxis. We furthermore demonstrate that activation triggers pertussis toxin-sensitive MAP kinase phosphorylation, which is also inhibited by GSK682753A. Thus, EBI2 signalling in B cells mediates key phenotypic functions via signalling pathways amenable to manipulation providing additional therapeutic options for inhibiting EBI2 activity.

Ligand modulation of the Epstein-Barr virus-induced seven-transmembrane receptor EBI2: identification of a potent and efficacious inverse agonist.

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6-53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe(111) at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC(50). In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.

Flow cytometry and other techniques show that Staphylococcus aureus undergoes significant physiological changes in the early stages of surface-attached culture.

The techniques of flow cytometry, scanning and transmission electron microscopy, and confocal scanning laser microscopy were used to study the physiology of Staphylococcus aureus in the early stages of surface-attached culture, and to make direct comparisons with planktonic bacteria grown under the same conditions. Attached bacteria growing in nutrient-rich batch culture were found to go through the same growth phases as equivalent planktonic cultures, but with an exponential growth rate of about half that of the planktonic bacteria. Viability of attached bacteria was very high (around 100%) throughout the first 24 h of growth. The size and protein content of attached bacteria varied with growth phase, and both measurements were always smaller than in planktonic bacteria at equivalent growth phases. Respiratory activity per bacterium, as measured by flow cytofluorimetry, and corrected for cell volume, peaked very early in attached cultures (before the first cell division) and declined from then on, whereas in planktonic bacteria it peaked in late exponential phase. Attached and planktonic bacteria showed thicker cell walls in stationary phase than in exponential phase. Membrane potentials of planktonic and attached bacteria were similar in stationary phase, but were much lower in exponential-phase attached cells than in the equivalent planktonic cells. It is apparent that a range of significant physiological adaptations occur during the early phases of attached growth.