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Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α 2A -adrenergic receptor (α 2A AR), seeking new α 2A AR agonists chemotypes that lack the sedation conferred by known α 2A AR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential G i and G o signaling. Experimental structures of α 2A AR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit ‘9087 [mean effective concentration (EC 50 ) of 52 nanomolar] and two analogs, ‘7075 and PS75 (EC 50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.KEYWORDS white-opaque switching; transcriptional regulation; transcription networks; transcriptional circuits; Candida albicans T HE fungal species Candida albicans is normally a harmless component of the human microbiome, but it can also cause life threatening bloodstream infections, particularly in immunocompromised patients. C. albicans has the unusual ability to switch between two cell types, named "white" and "opaque," each with distinct cellular and colony morphologies ( Figure 1, A and B) (Slutsky et al. 1987;Soll et al. 1993;Johnson 2003;Lohse and Johnson 2009;Soll 2009;Morschhäuser 2010). Each cell type is heritable for many cell divisions, and switching from one form to the other occurs without any change in the primary DNA sequence of the genome. Under standard laboratory conditions, switching between the two cell types occurs stochastically with approximately one switching event every 10 4 cell divisions, although certain environmental cues, such as elevated temperatures, can cause mass switching from one cell type to the other (Rikkerink et al. 1988;Huang et al. 2009Huang et al. , 2010. Expression of 20% of the genes in C. albicans is differentially regulated between the two cell types (Lan et al. 2002;) and, as a result, the white and opaque forms differ in their ability to mate (Miller and Johnson 2002), their metabolic preferences (Lan et al. 2002), and their interactions with the innate immune system (Kvaal et al. 1997(Kvaal et al. , 1999Geiger et al. 2004;Lohse and Johnson 2008;Sasse et a...

Peripheral nerve injury-induced neuropathic pain is a chronic and debilitating condition characterized by mechanical hypersensitivity. We previously identified microglial activation via release of colony stimulating factor 1 (CSF1) from injured sensory neurons as a mechanism contributing to nerve injury-induced pain. Here we show that intrathecal administration of CSF1, even in the absence of injury, is sufficient to induce pain behavior, but only in male mice. Transcriptional profiling and morphologic analyses after intrathecal CSF1 showed robust immune activation in male but not female microglia. CSF1 also induced marked expansion of lymphocytes within the spinal cord meninges, with preferential expansion of regulatory T-cells (Tregs) in female mice. Consistent with the hypothesis that Tregs actively suppress microglial activation in females, Treg deficient (Foxp3DTR) female mice showed increased CSF1-induced microglial activation and pain hypersensitivity equivalent to males. We conclude that sexual dimorphism in the contribution of microglia to pain results from Treg-mediated suppression of microglial activation and pain hypersensitivity in female mice.

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