Cell-to-Cell Transcription Variability as Measured by Single-Molecule RNA FISH to Detect Epigenetic State Switching

Single-molecule RNA fluorescent in situ hybridization (smRNA FISH) allows for the visualization, localization, and quantification of RNA transcripts within individual cells and tissues using custom-designed fluorescently labeled oligonucleotide probes. Here we describe a protocol for the preparation, imaging, and analysis of a smRNA FISH experiment that can be applied to any RNA of choice. We also provide insights as to how this powerful tool can be used to study epigenetic regulation, for example, following the epigenetic editing of genes

Reciprocal effects of interleukin-4 and interferon-γ on immunoglobulin E-mediated mast cell degranulation: a role for nitric oxide but not peroxynitrite or cyclic guanosine monophosphate

We report that cultured rat peritoneal cells spontaneously synthesize nitric oxide and this is associated with active suppression of mast cell secretory function. Addition of interleukin-4 (IL-4) or the nitric oxide synthase inhibitor N-monomethyl-l-arginine to peritoneal cells inhibited nitric oxide synthesis and enhanced anti-IgE-mediated mast cell degranulation, measured as serotonin release. Interferon-γ (IFN-γ) completely overcame the enhancement of serotonin release and suppression of nitrite production induced by IL-4. Over several experiments, with or without IL-4 and/or IFN-γ, serotonin release correlated inversely with nitrite production. On a cell-for-cell basis, non-mast cells produced ≈30 times more nitrite than mast cells in peritoneal cell populations, with or without IFN-γ stimulation. The nitric oxide donor S-nitrosoglutathione inhibited anti-IgE-induced serotonin release from purified mast cells, whereas 8-bromo-cyclic GMP, the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, superoxide dismutase and the peroxynitrite scavenger uric acid, were without effect. We conclude that IL-4 and IFN-γ reciprocally regulate mast cell secretory responsiveness via control of nitric oxide synthesis by accessory cells; the nitric oxide effect on mast cells is direct but does not involve cyclic GMP or peroxynitrite

Twinning in human populations and in cattle exposed to air pollution from incinerators

Human populations and animals are often exposed to the airborne pollutants in plumes from incinerators. The incineration of chemical and other waste may release polychlorinated hydrocarbons, some of which have oestrogenic properties. Increased numbers of twins had been reported anecdotally in cattle at risk from plumes from two incinerators near the town of Bonnybridge in central Scotland and also in cattle near a chemical factory in Eire. It was decided to follow up these reports in central Scotland and also to test the hypothesis that the frequency of human twinning might be increased there. Data on human twin and single births in hospitals in central Scotland were obtained for the years 1975-83. The twinning rates in areas exposed to airborne pollution from incinerators were compared with the background rates present in neighbouring areas. Farmers provided information on calving among the herds of two farms close to the incinerators. The frequency of human twinning was increased, particularly after 1979, in the areas most at risk from air pollution from the incinerators. Among the dairy cattle, there was a dramatic increase in twinning at about the same time