Rep1p is required to down-regulate a subset of nitrogen starvation-induced genes

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulatory network for sexual differentiation in fission yeast"</p><p>http://genomebiology.com/2007/8/10/R217</p><p>Genome Biology 2007;8(10):R217-R217.</p><p>Published online 10 Oct 2007</p><p>PMCID:PMC2246291.</p><p></p> Average expression profiles in wild-type (synchronized cells from [4]) and cells of two subclusters of genes induced in response to nitrogen starvation: delayed (blue) and transient (red), as defined by [4]. Hierarchical cluster analysis of the two gene clusters shown in (a), with columns representing experimental time points and rows representing genes. The mRNA levels at each time point of time courses relative to the levels in vegetative cells are color-coded as indicated at the bottom with missing data in gray. Labeling is as in Figure 1. Histogram showing the gene expression levels of Δ relative to wild-type meiotic cells. The two clusters are colored as in (a)

DS_DISC764678 – Supplemental material for Plate-Based Phenotypic Screening for Pain Using Human iPSC-Derived Sensory Neurons

<p>Supplemental material, DS_DISC764678 for Plate-Based Phenotypic Screening for Pain Using Human iPSC-Derived Sensory Neurons by Peter Stacey, Anne Mai Wassermann, Laura Kammonen, Emma Impey, Anna Wilbrey and Darren Cawkill in SLAS Discovery</p

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release

<div><p>Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.</p></div

PF-05198007 reduces the capsaicin flare response in WT, but not Na_v1.7^Nav1.8Cre mice.

<p>A, B.Time-course plots showing the effects of PF-05198007 on skin blood flow measured before and after topical capsaicin application for WT (A) and Nav1.7^Nav1.8Cre (B) mice (for each genotype, n = 8 per group). C, D. Corresponding summary bar graphs showing flare response measured as area under the curve for WT (C) and Nav1.7^Nav1.8Cre (D) mice before and after PF-05198007 treatment. 1 mg/kg and 10 mg/kg PF-05198007 significantly reduced capsaicin-induced flare in WT mice (C, both 1 mg/kg and 10 mg/kg, p < 0.01, ANOVA) but had no effect in Na_v1.7^Nav1.8Cre mice (D, both 1 mg/kg and 10 mg/kg, p > 0.05, ANOVA).</p

Potency of PF-05089771 across hNa_v1.7 splice variants.

<p>Potency of PF-05089771 across hNa_v1.7 splice variants.</p

PF-05089771 is a potent, state-dependent and selective inhibitor of Na_v1.7.

<p>A. Structure of PF-05089771 (4-(2-(3-amino-1H-pyrazol-4-yl)-4-chlorophenoxy)-5-chloro-2-fluoro-N-(thiazol-4-yl)benzenesulfonamide) B. Representative PatchXpress current recordings illustrating the near-complete block following 300 nM PF-05089771 application to half-inactivated WT hNa_v1.7 channels (97% ± 3%, n = 10) which was partially reversed following a 5 min washout duration. In contrast there was minimal block following application of 300 nM PF-05089771 to resting WT hNa_v1.7 channels (5% ± 3%, n = 4). Inset: PatchXpress voltage protocols for half-inactivation (upper) and resting state (lower). For a full description of the voltage protocols see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#sec002" target="_blank">Methods</a>. C. Block of half-inactivated WT hNa_v1.7 channels (n = 6–22 per concentration) was nearly 1000-fold more potent than resting channels (n = 4–11 per concentration) (11 nM <i>vs</i> 10 μM). D. Potency of PF-05089771 was similar across hNa_v1.7 splice variants. IC₅₀ values and Hill slopes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t001" target="_blank">Table 1</a>. Data points represent n = 2–9 observations per concentration. E. PF-05089771 activity is impacted by mutation of a novel interaction site and not by local anaesthetic or toxin binding sites. Data points represent n = 3–6 observations per concentration except for hNa_v1.7 where n = 6–22 observations per concentration. F. Potency of PF-05089771 was assessed on orthologous channels cloned from common preclinical species. IC₅₀ values and Hill slopes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t002" target="_blank">Table 2</a>. Data points represent n = 2–28 observations per concentration. G. PF-05089771 is a selective Na_v1.7 subtype-selective inhibitor. Selectivity was assessed across a collection of heterologously expressed human Na_vs on PatchXpress at the unique half inactivation voltage for each channel. Hill slopes and IC₅₀ values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152405#pone.0152405.t003" target="_blank">Table 3</a>. Data points represent n = 3–12 observations per concentration except for hNa_v1.7 where n = 6–22 observations per concentration. Selectivity over the TTX-R Na_v1.5 and Na_v1.8 channels was greater than 1000-fold.</p

Na_v1.7 is the major TTX-sensitive Na_v channel in small diameter mDRG neurons.

<p>A. RNASeq analysis of Na_v channel mRNA from pooled small diameter mouse DRG neurons. B. Structure of PF-05198007 (4-(2-(3-amino-1H-pyrazol-4-yl)-4-(trifluoromethyl)phenoxy)-5-chloro-2-fluoro-N-(thiazol-4-yl)benzenesulfonamide C. Patch clamp data showing concentration-response relationship for PF-05198007 assessed against recombinantly expressed mouse Na_v1.7, Na_v1.6 and Na_v1.1 (IC₅₀, Slope: 5.2 nM, 1.1; 149 nM, 1.5; 174 nM, 0.7 respectively; n = 3–4 per concentration). D. Representative patch clamp current traces of peak sodium current from small diameter mouse DRG neurons in the presence of A-803467 and following concurrent application of PF-05198007 and TTX. E. Representative peak TTX-S current <i>vs</i> time plot before and after 30 nM PF-05198007 and 500 nM TTX. G. Scatter plot of cell capacitance <i>vs</i> Na_v1.7/TTX-S ratio (n = 35). Note that in every cell tested, Na_v1.7 provided the predominant TTX-S sodium conductance.</p

PF-05198007 increases action potential rheobase in small diameter mDRG neurons.

<p>A. Overlayed representative voltage traces in response to graded current step injections before (blue) and after PF-05198007 application (red). Current step stimulations are shown below. B. Example timecourse of change in rheobase following PF-05198007 application and washout. C. Summary bar graph, n = 8 neurons, ** p < 0.01, ANOVA. Data are shown ±SEM.</p

Potency of PF-05089771 assessed at orthologous channels from selected species.

<p>Potency of PF-05089771 assessed at orthologous channels from selected species.</p

Evidence for functional Na_v1.7 in human DRG neurons.

<p>A. Representative TTX-S current traces (recorded in the presence of 1 μM A-803467 and following graded voltage steps from -110 mV to 10 mV. B. Voltage dependence of activation (red, n = 4 for each voltage) generated from the protocol described in A and steady state fast inactivation (blue) generated by conditioning 500 msec prepulses to voltages between -110 mV and +10 mV followed by a test pulse to 0 mV from a holding potential of -110 mV (n = 4 for each voltage). Both datasets are fitted with Boltzmann functions. C. Representative timecourse relationship for peak TTX-S current following the application of 100 nM PF-05089771 and 500 nM TTX. D. Concentration-response relationship for PF-05089771 block of TTX-S current (IC₅₀, slope: 8.4 nM, 1.1; n = 3–6 per concentration) E. Example voltage traces from a current clamp recording. Single action potentials were evoked by a 20 ms suprathreshold current step at 0.1 Hz. The scale bar refers to the voltage traces whereas the start-to-start interval is 10 s. F. Summary pie charts showing that the application of 30 and 100 nM PF-05089771 resulted in action potential block in 3/7 and 5/8 DRG neurons respectively.</p