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Systemic administration of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, removes transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons and impairs thermal but not mechanical nociception in adult animals. In this study, we determined how loss of TRPV1-expressing sensory neurons alters the antinociceptive effect of μ opioids and μ opioid receptors in the spinal cord. The effect of morphine and (d-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) was measured by testing the paw mechanical withdrawal threshold in rats treated with RTX or vehicle. RTX treatment deleted TRPV1-immunoreactive dorsal root ganglion neurons and nerve terminals in the spinal dorsal horn. Also the μ opioid receptor immunoreactivity was markedly reduced in the superficial dorsal horn of RTX-treated rats. However, RTX treatment did not affect the dorsal horn neurons labeled with both TRPV1- and μ opioid receptor-immunoreactivity. Surprisingly, intrathecal morphine or DAMGO produced a greater increase in the withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the effect of intrathecal morphine and DAMGO in RTX-treated rats was also profoundly increased. Furthermore, the antinociceptive effect of systemic morphine was significantly potentiated in RTX-treated rats. The BMAX (but not KD) of [3H]-DAMGO binding and DAMGO-stimulated [35S]GTPγS activity in the dorsal spinal cord were significantly reduced in the RTX group. This study provides novel information that loss of TRPV1 afferent neurons eliminates presynaptic μ opioid receptors present on TRPV1-expressing afferent neurons but paradoxically potentiates the analgesic effect of μ opioid agonists. Mechano-nociception, transmitted through non-TRPV1 sensory neurons, is subject to potent modulation by μ opioid agonists.
Nociception is transduced by different populations of primary sensory neurons and appears modality specific. The small-diameter dorsal root ganglion (DRG) neurons and associated Aδ- and C-fiber afferents are critical for detecting noxious stimuli and initiating pain sensation (Burgess and Perl 1967; Harper and Lawson 1985; Slugg et al. 2000). These DRG neurons are functionally and phenotypically diverse and express various receptors and ion channels. The capsaicin receptor, transient receptor potential vanilloid type 1 (TRPV1), plays an essential role in detecting thermal nociception. In this regard, mice deficient of TRPV1 show impaired pain response to heat (Caterina et al. 2000). Resiniferotoxin (RTX), originally isolated from the cactus-like plant Euphorbia resinifera, is an ultrapotent TRPV1 agonist (Szallasi and Blumberg 1999). Systemic injection of RTX destroys TRPV1-expressing sensory neurons and induces a long-lasting impairment of thermal nociception in adult rats (Pan et al. 2003; Xu et al. 1997). Interestingly, mechano-nociception is largely intact in RTX-treated rats (Pan et al. 2003; Xu et al. 1997). Thus RTX is a valuable tool to study the nociceptive pathway transmitted through non-TRPV1 afferent neurons and its modulation.
The μ opioid receptor agonists, such as morphine, remain the most potent analgesics to treat patients with moderate to severe pain. Opioids have a distinct effect on nociception with little effect on other sensory modalities. The spinal cord dorsal horn is critically involved in pain transmission and modulation and is a major site for the analgesic action of μ opioid agonists (Chen et al. 2005; Magnuson and Dickenson 1991; Yaksh and Noueihed 1985). The μ opioid receptors are located presynaptically on central terminals of primary afferent neurons as well as postsynaptic dorsal horn neurons (Fields et al. 1980; Gamse et al. 1979; Yoshimura and North 1983). However, the relative role of pre- and postsynaptic μ opioid receptors in the spinal opioid analgesia is not fully known.
In addition to the loss of TRPV1-expressing afferent neurons, RTX may eliminate μ opioid receptors located on the TRPV1-expressing presynaptic terminals in the spinal dorsal horn. The influence of destruction of TRPV1-expressing afferent neurons on the antinociceptive effect of opioids has not been studied previously. Therefore we used RTX-treated animals to determine the relative importance of presynaptic μ opioid receptors in spinal opioid analgesia. Unexpectedly, we found that although destruction of TRPV1-expressing afferent neurons eliminates presynaptic μ opioid receptors present on TRPV1-expressing afferent neurons, it paradoxically potentiates the antinociceptive effect produced by intrathecal and systemic μ opioid agonists. This study reveals an important function of μ opioid receptors in modulation of mechano-nociception transmitted by non-TRPV1 afferent neurons and extends our current knowledge of the pain pathways and mechanisms underlying opioid analgesia.
Male rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 200–220 g were used in this study. Rats received a single intraperitoneal injection of RTX (200 μg/kg, LC Laboratories, Woburn, MA) under halothane (2% in O2) anesthesia. RTX was dissolved in a mixture of 10% Tween-80 and 10% ethanol in normal saline (Pan et al. 2003). Rats in the control group received intraperitoneal injection of the vehicle. The experiments were conducted 4–5 wk after RTX and vehicle injections unless stated otherwise. The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine and conformed to National Institutes of Health guidelines on the ethical use of animals.
Intrathecal catheters (PE-10 polyethylene tubing) were inserted in RTX- and vehicle-treated rats during halothane-induced anesthesia. The catheters were advanced 8 cm caudal through an incision in the cisternal membrane and secured to the musculature at the incision site. The rats were allowed to recover for 5–7 days before being used to test the antinociceptive effect of morphine or (d-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO). Only animals with no evidence of neurological deficits after catheter insertion were studied. Drugs for intrathecal injection were dissolved in normal saline and administered in a volume of 5 μl followed by a 10 μl flush with normal saline. Repeat intrathecal injections in the same animals were separated by 4–5 days. Morphine was obtained from Astra Pharmaceuticals (Westborougn, MA). DAMGO and d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were purchased from Sigma (St. Louis, MO).
Behavioral assessment of thermal nociception
To quantitatively assess the thermal sensitivity of the normal rats, rats were placed on the glass surface of a thermal testing apparatus (IITC, Woodland Hills, CA). The rats were allowed to acclimate for 30 min before testing. The temperature of the glass surface was maintained constant at 30°C. A mobile radiant heat source located under the glass was focused onto the hindpaw of each rat. The paw-withdrawal latency was recorded by a timer, and the mean value from both hindpaws was used. The cut-off of 30 s was used to prevent potential tissue damage (Chen and Pan 2003a; Pan et al. 2003).
Behavioral testing of mechanical nociception
The nociceptive mechanical threshold was measured using an Ugo Basil Analgesimeter (Varese, Italy). The test was performed by applying a noxious pressure to the hindpaw. By pressing a pedal that activated a motor, the force increased at a constant rate on the linear scale. When the animal displayed pain by withdrawal of the paw or vocalization, the pedal was immediately released and the nociceptive threshold read on a scale. The cut-off of 400 g was used to avoid potential tissue injury (Chen and Pan 2003b). Both hindpaws were tested in each rat, and the mean value was used as the nociceptive withdrawal threshold.
Double fluorescence labeling of TRPV1 and IB4 in DRGs and spinal dorsal horn
To determine the effect of RTX treatment on TRPV1-positive and Griffonia simplicifolia isolectin B4 (IB4)-positive DRG neurons and central terminals, double labeling of TRPV1 and IB4 [a marker for unmyelinated afferent fibers (Kitchener et al. 1993, 1994)] in DRG neurons and the spinal dorsal horn was performed on three vehicle- and four RTX-treated rats 4 wk after treatment. Under deep anesthesia with pentobarbital sodium (60 mg/ml ip), each rat was intracardially perfused with 250 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) and 200 ml of 10% sucrose in 0.1 M PBS (pH 7.4). The lumbar spinal cord and L4 and L5 DRGs were removed quickly and postfixed in the same fixative solution and cryoprotected in 30% sucrose in PBS for 48 h at 4°C. The tissues were cut to 30 μm in thickness and collected free-floating in 0.1 M PBS. For TRPV1 immunofluorescent labeling, the sections were rinsed in 0.1 M PBS and blocked in 4% normal goat serum in PBS for 1 h. The sections were then incubated with the primary antibody (rabbit anti-TRPV1 N-terminus, dilution 1:1000, Neuromics, Minneapolis, MN) diluted in PBS containing 2% normal goat serum and 0.3% TX-100 for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed in PBS and incubated with the secondary antibody (Alexa Fluor-488 conjugated to goat anti-rabbit IgG, dilution: 5 μg/ml, Molecular Probes, Eugene, OR). The sections then were rinsed in PBS for 30 min and incubated with Alexa Fluor 594 conjugated to IB4 (Molecular Probes, dilution: 2 μg/ml) for 2 h at room temperature. Finally, the sections were rinsed and mounted on slides, dried, and coverslipped. The sections were examined on a laser scanning confocal microscope (Leica, Germany), and areas of interest were photodocumented.
Double immunofluorescence labeling of TRPV1 and μ opioid receptors in the DRG and spinal cord
To examine the effect of RTX treatment on the distribution of μ opioid receptors in the DRGs and spinal dorsal horn, double immunofluorescence labeling of μ opioid receptors and TRPV1 was performed in L4 and L5 DRGs and the lumbar spinal cord from three RTX- and three vehicle-treated rats. As described previously (Chen and Pan 2003a; Wu et al. 2004, 2005), the tissue sections were rinsed in 0.1 M PBS and blocked in 4% normal goat serum in PBS for 1 h. Then sections were incubated with the mixture of primary antibodies (rabbit anti-μ opioid receptors, dilution 1:1000, Neuromics; guinea pig anti-TRPV1, dilution 1:1,000, Neuromics) diluted in PBS solution containing 2% normal goat serum and 0.3% TX-100 for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed in PBS and incubated with the secondary antibody mixture (Alexa Fluor 488 conjugated to goat anti-rabbit IgG, Alexa Fluor 594 conjugated to goat anti-guinea pig IgG, Molecular Probes, dilution: 5 μg/ml) for 1.5 h. The sections then were rinsed in PBS for 30 min, mounted on slides, dried, and coverslipped. The sections were examined on a laser scanning confocal microscope. Omission of the primary antibody and preabsorption of the anti-μ opioid receptor antibody with the blocking peptide resulted in negative labeling in the DRGs and the spinal cord. The cell counting of DRG neurons was performed by an investigator who was blinded to the experimental condition.
[3H]-DAMGO membrane bindings in the spinal cord
To determine if RTX treatment alters the binding number and affinity of the μ opioid receptors in the spinal cord, saturation binding of [3H]-DAMGO, a specific radioligand for μ opioid receptors, was carried out using the spinal cord tissue membranes (Chen and Pan 2003a). Six RTX- and six vehicle-treated rats were decapitated after being anesthetized with halothane. The whole spinal cord was quickly harvested, and the dorsal half was dissected and used for the binding experiment. The tissue was homogenized in ice-cold 50 mM Tris buffer containing 3 mM MgCl2 and 1 mM EGTA (pH 7.4) and disrupted by sonication. The homogenate was then centrifuged at 500 g for 10 min at 4°C. The pellet was discarded and the supernatant was centrifuged at 48,000 g for 20 min at 4°C. The pellet was resuspended in fresh Tris buffer and was centrifuged again as described in the preceding text. The final pellet was resuspended in 50 mM Tris buffer containing (in mM) 3 MgCl2, 100 NaCl, and 0.2 EGTA (pH 7.7) and disrupted by sonication for 5 s. Saturation radioligand binding experiments were performed using 200 μl aliquots of tissue and increasing concentrations of [3H]-DAMGO (76 Ci/mmol, Amersham Biosciences, Piscataway, NJ) from 0.3 to 11 nM, as we described previously (Chen and Pan 2003a). Nonspecific binding was determined with 1 μM naloxone (Sigma, St. Louis, MO). Incubation was performed in duplicate in Tris buffer at 25°C for 60 min. The reaction was terminated by filtration through Whatman GF/B filters on a cell harvester with cold Tris buffer (pH 7.4). Radioactivity was quantified by immersion of filters in the scintillation fluid, then incubated overnight at room temperature, and measured the next day using a liquid scintillation counter (LS 6500, Beckman Coulter, Fullerton, CA). The protein content was measured based on the method of Bradford using the bovine serum albumin as standards (Protein Assay Kit II, Bio-Rad Laboratories, Hercules, CA).
DAMGO [35S]GTPγS activity in spinal cord membranes
Additional experiments were performed to further determine if the functional coupling of μ opioid receptors to G proteins is altered by RTX treatment. The [35S]GTPγS binding assay is based on the principle that the inactive state of the G protein α subunit has a relatively high affinity for GDP over GTP, whereas activation of a G-protein-coupled receptor by its agonist shifts the α subunit into a higher affinity for GTP versus GDP. The [35S]GTPγS is a hydrolysis-resistant form of GTP, and the degree to which an agonist stimulates [35S]GTPγS activity can be quantified in tissue membranes. Thus the agonist-stimulated [35S]GTPγS binding can provide functional information about the receptor (i.e., coupling of G proteins to activated receptors). As described previously (Chen et al. 2002), the DAMGO-stimulated [35S]GTPγS binding was performed using isolated membranes. The dorsal spinal cord tissue was homogenized in cold membrane buffer [containing (in mM) 50 Tris-HCl, 3 MgCl2, and 1 EGTA, pH 7.4] and centrifuged at 500 g for 10 min at 4°C. The supernatant was centrifuged at 48,000 g for 30 min at 4°C. The pellet was resuspended in assay buffer [containing (in mM) 50 Tris-HCl, 3 MgCl2, 0.2 EGTA, and 100 NaCl, pH 7.7] and measured for protein content. Concentration-effect curves of the DAMGO-stimulated [35S]GTPγS binding included 0.1–10 μM DAMGO with and without 1 μM d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP, a specific μ opioid receptor antagonist), 30 μM GDP, 0.05 nM [35S]GTPγS, 4 mU/ml adenosine deaminase, 20 μg protein, and assay buffer in a final volume of 1 ml. The basal binding was determined in the presence of GDP and absence of agonists, and the nonspecific binding was assessed in the presence of 10 μM GTPγS. After incubation for 1 h at 30°C, reactions were terminated by rapid filtration (Brandel, Gaithersburg, MD) through Whatman GF/B glass fiber filters followed by three washes with 3 ml cold Tris buffer (50 mM Tris-HCl, pH 7.7). Bound radioactivity was determined by the liquid scintillation counter after overnight extraction of the filter in 7 ml scintillation fluid.
Statistical analysis
Data are presented as means ± SE. Paw-withdrawal thresholds in response to thermal and mechanical stimulation before and after RTX treatment were compared using a paired Student's t-test or repeated-measures ANOVA. The effect of morphine and DAMGO on the mechanical withdrawal threshold was determined by repeated measures followed by Tukey's post hoc test. Because the strongest component of potentiation of opioid action in this study was the extended duration of opioids, the ED50 value was calculated by integrating the area under the time-effect curve for each rat. The [3H]-DAMGO saturation binding data were processed using nonlinear regression analysis (Prism; GraphPad Software, San Diego, CA) to calculate maximal specific binding (Bmax) and dissociation constant (KD). For DAMGO-stimulated [35S]GTPγS binding, nonlinear regression analyses of concentration-effect curves were also performed using Prism. The percent of stimulation of [35S]GTPγS activity was calculated as: (net stimulated binding/basal binding) X 100%. Differences were considered to be statistically significant when P < 0.05.
Effect of RTX on the withdrawal threshold in response to noxious heat and pressure stimuli
Systemic injection of RTX caused a large increase in the paw-withdrawal threshold in response to the heat stimulus (Fig. 1A). This diminished thermal sensitivity occurred within 3 days after RTX injection and lasted for the duration (5 wk) of the experiments. Furthermore, RTX produced a small, but significant, decrease in the paw-withdrawal threshold in response to noxious pressure applied to the hindpaw (Fig. 1B). This slight increase in mechanical sensitivity also appeared within 3 days after RTX injection and remained unchanged for ≥5 wk. By contrast, systemic injection of the vehicle did not significantly alter the paw-withdrawal threshold in response to the noxious heat and pressure stimuli during the entire period of the experiment (Fig. 1, A and B).
Effect of RTX on IB4-labeling and TRPV1-immunoreactive DRG neurons and afferent fibers in the spinal dorsal horn
To examine the effect of RTX treatment on TRPV1- and IB4-positive primary afferent neurons, double fluorescent labeling was conducted in the L4 and L5 DRG from vehicle- and RTX-treated rats 4 wk after treatment. TRPV1 immunoreactivity and IB4-labeling were present in small- and medium-sized DRG neurons in vehicle-treated rats (Fig. 2A). IB4-labeling was present in most TRPV1-immunoreactive neurons. In contrast, the TRPV1-immunoreactive cell bodies were largely absent in DRGs removed from RTX-treated rats (Fig. 2A). IB4-positive neurons were also substantially reduced in DRGs of RTX-treated rats. To quantify the reduction of TRPV1- and IB4-positive DRG neurons by RTX, six confocal images were randomly selected from each DRG (two DRGs/rat) in three RTX- and three vehicle-treated rats, and the total number of TRPV1-immunoreactive and IB4-positive cell bodies was counted from each section. This analysis showed that ∼97% (1034 in vehicle control vs. 31 in RTX group) TRPV1-immunoreactive DRG neurons were lost in RTX-treated rats. Furthermore, there was a 68% (1189 in vehicle control vs. 381 in RTX group) reduction in IB4-positive DRG neurons in RTX-treated animals.
In vehicle-treated rats, TRPV1-immunoreactive- and IB4-positive terminals were present in the superficial dorsal horn of the spinal cord (Fig. 2B). As illustrated in Fig. 2B, dense TRPV1 immunoreactivity was seen both at the dorsal root entry zone and in laminas I and II. TRPV1 immunoreactivity appeared largely co-localized on the same terminals labeled with IB4 in the laminas I and II of rats treated with the vehicle. By comparison, the TRPV1-immunoreactive terminals were completely abolished in the spinal cord dorsal horn in RTX-treated rats (Fig. 2B). Also, IB4-positive terminals were markedly decreased in the spinal dorsal horn of RTX-treated rats. This decrease was especially prominent in the more medial region of the dorsal horn (Fig. 2B). The reduction in IB4-positive afferent terminals and the depletion of TRPV1 immunoreactivity in the superficial dorsal horn of RTX-treated rats both reflect the cytotoxic effect of RTX on a subpopulation of sensory neurons. These findings indicate that RTX treatment destroys TRPV1-expressing afferent neurons and their central terminals in the spinal dorsal horn.
Effect of RTX treatment on the immunoreactivity of μ opioid receptors and TRPV1 in DRG neurons and the spinal dorsal horn
As shown in Fig. 3A, the μ opioid receptor immunoreactivity was present in different sizes of DRG neurons in vehicle-treated rats. Also, small-diameter DRG neurons contained immunoreactivity for both TRPV1 and μ opioid receptors (Fig. 3A). Furthermore to quantify the reduction of the μ opioid receptor immunoreactive DRG neurons by RTX, the total number of μ opioid receptor-immunoreactive cell bodies was counted from six confocal images randomly selected from each DRG (2 DRGs/rat) in three RTX- and three vehicle-treated rats. This analysis showed that ∼27% (1,548 in vehicle control vs. 1,130 in RTX group) decrease in the number of cell bodies immunoreactive to the μ opioid receptors in RTX-treated rats (Fig. 3A).
The immunoreactivity of μ opioid receptors was mainly concentrated in the superficial laminas of the spinal dorsal horn in vehicle-treated rats (Fig. 3B). The μ opioid receptor immunoreactivity appeared co-localized on some terminals labeled with TRPV1 in laminas I and II of the spinal cord (Fig. 3B). In RTX-treated rats, TRPV1 immunoreactivity was largely eliminated in the superficial dorsal horn. There was also a substantial reduction in the density of μ opioid receptor immunoreactivity in the laminas I and II of the spinal cord of RTX-treated rats (Fig. 3B). Furthermore, a subpopulation of dorsal horn neurons containing TRPV1 immunoreactivity was present in the superficial dorsal horn, and these neurons were also immunoreactive to μ opioid receptors (Fig. 4A). Interestingly, the dorsal horn neurons that contained both μ opioid receptor and TRPV1 immunoreactivities were not affected by RTX treatment (Fig. 4B).
Effect of morphine and DAMGO on mechanical nociception in RTX- and vehicle-treated rats
Because RTX treatment removed μ opioid receptor immunoreactivity present in TRPV1-expressing DRG neurons and afferent terminals in the spinal cord, we initially tested the effect of intrathecal morphine on the hindpaw-withdrawal threshold in response to the noxious pressure in RTX- and vehicle-treated rats 4 wk after treatment. Intrathecal injection of 2–10 μg morphine in the vehicle group dose-dependently increased the withdrawal threshold in response to the pressure stimulus (Fig. 5A). The maximal effect of morphine appeared within 30 min after intrathecal injection, and the effect gradually diminished within 2–3 h. By comparison, intrathecal injection of 2–10 μg morphine produced a large effect on the pressure-evoked paw-withdrawal threshold in RTX-treated rats (Fig. 5B). Notably, 5 and 10 μg of morphine increased the withdrawal threshold to the cut-off values, and this effect lasted for 5–6 h after a single intrathecal injection in RTX-treated rats (Fig. 5B). The estimated ED50 value (95% confidence limits) in vehicle- and RTX-treated groups was 4.2 (2.3–12.6) and 1.1 (0.06–1.9) μg, respectively (P < 0.05). Similar potentiated and prolonged effect of intrathecal morphine was also observed in seven separate rats tested 10–14 days after RTX injection (data not shown). Intrathecal pretreatment with a highly specific μ opioid receptor antagonist, CTAP (3 μg), 15 min before morphine administration abolished the effect of intrathecal injection of 10 μg morphine on the paw-withdrawal threshold (Fig. 5B). Intrathecal injection of 3 μg CTAP alone had no significant effect on the withdrawal threshold in all rats tested (data not shown).
We next compared the effect of a highly specific μ opioid peptide agonist, DAMGO, on the paw-withdrawal threshold in RTX- and vehicle-treated rats. Intrathecal injection of 0.1 and 0.3 μg DAMGO significantly increased the withdrawal threshold in response to the pressure stimulus, and this effect lasted ∼45 min in the vehicle group (n = 8, Fig. 6). By comparison, the same doses of DAMGO produced a significantly larger increase in the withdrawal threshold in the RTX group (Fig. 6). Intrathecal injection of 0.3 μg DAMGO elevated the withdrawal threshold to the cut-off for ∼45 min after injection in RTX-treated rats.
To determine if loss of TRPV1-expressing afferent neurons alters the antinociceptive effect of the μ opioid given systemically, we further tested the effect of intraperitoneal administration of 2.5 and 5 mg/kg morphine in additional RTX- and vehicle-treated rats. In the vehicle group, intraperitoneal injection of 2.5 and 5 mg/kg morphine significantly increased the hindpaw-withdrawal threshold in response to the pressure stimulus (Fig. 7). Interestingly, the same doses of morphine given intraperitoneally produced a larger and longer increase in the paw-withdrawal threshold in the RTX than in the vehicle group (Fig. 7).
[3H]-DAMGO saturation binding in the spinal cord of RTX- and vehicle-treated rats
To determine if RTX treatment changes the number of binding sites and affinity of μ opioid receptors in the spinal cord, we analyzed [3H]-DAMGO saturation binding using membranes of the dorsal spinal cord from RTX- and vehicle-treated rats. In these experiments, membrane homogenates were prepared from individual rats (n = 6 rats in each group), and six replicate experiments were performed. We observed that [3H]-DAMGO had a single and saturable high-affinity binding site in the dorsal spinal cord tissue in both the RTX and vehicle groups (Fig. 8A). The maximal specific binding (Bmax) of [3H]-DAMGO binding in RTX-treated rats was significantly reduced compared with that in control rats (178.6 ± 5.2 vs. 237.6 ± 5.4 fmol/mg protein, P < 0.05). However, the dissociation constant (KD) value was similar in the vehicle (1.26 ± 0.14 nM)- and RTX (1.23 ± 0.18 nM)-treated groups.
DAMGO [35S]GTPγS activity in the spinal cord membranes from RTX- and vehicle-treated rats
Additionally, to further examine if RTX treatment induced potential changes in the μ opioid receptor-G protein coupling in the spinal cord, the [35S]GTPγS activity stimulated by DAMGO was performed using isolated membranes from the dorsal spinal cord of RTX- and vehicle-treated rats. In these experiments, membrane homogenates were prepared from individual rats (n = 8 rats in each group), and eight replicate experiments were run. The concentration-effect curve for the DAMGO-stimulated [35S]GTPγS binding in spinal cord membranes is shown in Fig. 8B. The DAMGO-stimulated [35S]GTPγS binding was completely blocked by 1 μM CTAP (data not shown). Compared with that in vehicle-treated rats, the maximal DAMGO-stimulated [35S]GTPγS activity in the dorsal spinal cord was significantly reduced in RTX-treated rats (63.7 ± 2.5 vs. 48.5 ± 1.4%, P < 0.05, Fig. 8B). When [35S]GTPγS activity stimulated by 10 μM DAMGO was considered as the maximal response in each group, the estimated EC50 value (95% confidence limits) of DAMGO was 0.44 (0.14–2.45) and 0.46 (0.16–2.69) μM in the control and RTX group, respectively. The basal [35S]GTPγS binding was not significantly different between RTX- and vehicle-treated rats (56.6 ± 2.2 vs. 59.2 ± 2.4 fmol/mg protein, P > 0.05).
This is the first study to determine the influence of loss of TRPV1 sensory neurons and reduction of presynaptic μ opioid receptors on the spinal opioid analgesic effect. Because RTX deletes TRPV1-expressing primary afferent nerves, this will likely reduce the presynaptic μ opioid receptors localized on the central terminals of these neurons. This offers a unique opportunity to determine the relative role of presynaptic μ opioid receptors in analgesia produced by spinal μ opioids. In the present study, we demonstrated that removal of TRPV1-expressing afferent neurons by RTX not only failed to attenuate the antinociceptive effect of μ opioid agonists but unexpectedly caused a large potentiation of the antinociceptive effect of intrathecal and systemic μ opioid agonists. To our knowledge, this augmented analgesic effect of spinal μ opioid agonists after reduction of spinal presynaptic μ opioid receptors has not been reported previously.
The spinal cord dorsal horn is a critical site for the processing of nociceptive information and for the opioid analgesic effect (Dickenson 1995; Light and Willcockson 1999; Saeki and Yaksh 1993; Yaksh and Noueihed 1985). Opioids likely inhibit synaptic transmission through both pre- and postsynaptic mechanisms in the spinal dorsal horn. About 75% of μ opioid receptors in the spinal cord are located presynaptically on primary afferent terminals (Dickenson 1995). Activation of presynaptic μ opioid receptors reduces the release of excitatory neurotransmitters from the central terminals of primary afferent neurons and inhibits nociceptive inputs into the spinal cord dorsal horn (Chen et al. 2005; Kohno et al. 1999; Light and Willcockson 1999; Schneider et al. 1998). Thus the presynaptic site of action of μ opioid agonists on nociceptive afferent terminals in the spinal cord has been considered an important mechanism underlying spinal opioid analgesia. Because RTX selectively removes TRPV1-expressing afferent neurons and the associated presynaptic μ opioid receptors in the spinal cord, this study was originally designed to examine to what extent the analgesic effect of spinally administered μ opioid agonists would be attenuated in rats treated with RTX. There is no evidence suggesting that RTX acts through TRPV2 channels. RTX is a well-known capsaicin analogue, and its binding is competitively inhibited by TRPV1 antagonist capsazepine (Szallasi et al. 1999). Furthermore, in contrast to TRPV1, TRPV2 is expressed in medium- to large-sized DRG neurons with myelinated fibers and does not respond to capsaicin (Lewinter et al. 2004; Tamura et al. 2005). It is important to note that the effect of RTX is not to specifically eliminate the TRPV1 channels but rather to destroy the primary afferent neurons expressing TRPV1 channels. Consequently, it is likely that any ion channels and receptors that are present on TRPV1-expressing DRG neurons are also eliminated by RTX treatment. Destruction of TRPV1-expressing DRG neurons by the cytotoxicity of RTX is clearly supported by our finding showing that the number of μ opioid receptor-immunoreactive and IB4-positive DRG neurons was markedly reduced by RTX treatment. Furthermore it has been documented that both RTX and capsaicin can kill primary sensory neurons through calcium influx and overloading (Karai et al. 2004; Olah et al. 2001; Wu et al. 2005). Our immunocytochemistry data confirmed that RTX eliminated presynaptic μ opioid receptors on TRPV1-immunoreactive afferent terminals in the spinal dorsal horn. The radioligand binding experiments also demonstrated ∼30% reduction of the specific binding site of μ opioid receptors in the spinal cord of RTX-treated rats. It has been shown that TRPV1 is expressed by a subpopulation of dorsal horn neurons in laminas I and II (Doly et al. 2004; Valtschanoff et al. 2001). Interestingly, we found that systemic RTX treatment had no effect on the superficial dorsal horn neurons that express both TRPV1 and μ opioid receptors. Previous studies have shown that neonatal capsaicin treatment reduces μ opioid receptors in the spinal cord (Abbadie et al. 2002) but does not affect morphine analgesia in rats (Jancso and Jancso-Gabor 1980). In contrast to our hypothesis, we found that loss of TRPV1-expressing afferent neurons paradoxically potentiates the antinociceptive effect of μ opioid agonists. In this regard, intrathecal injection of two structurally dissimilar μ opioid receptor agonists, morphine and DAMGO, produced a profound effect on nociception measured with a noxious pressure stimulus in RTX-treated rats. Additionally, the analgesic effect produced by intraperitoneal morphine was also significantly potentiated in rats treated with RTX, suggesting that the μ opioid agonist administered systemically also is more efficacious in suppressing the mechano-nociception in animals lacking TRPV1-expressing afferent neurons.
RTX-induced paradoxical changes in the reduction of spinal μ opioid receptors and potentiation of the potency of opioid analgesia are completely unexpected. Changes in the μ opioid receptor number/binding affinity, redistribution, desensitization, and G protein coupling and downstream signaling may explain increased opioid efficacy in RTX-treated rats. Our immunocytochemistry and radioligand binding data do not support the possibility that the potentiated analgesic effect of spinal μ opioid agonists is due to the compensatory upregulation of μ opioid receptors in the spinal cord of RTX-treated rats. Both immunocytochemistry and radioligand-binding experiments showed a consistent reduction of μ opioid receptors in the spinal cord of RTX-treated rats. We also found that the KD value of the specific μ opioid receptor binding in the spinal cord was similar in the vehicle and RTX groups. Furthermore the DAMGO-stimulated [35S]GTPγS activity in the dorsal spinal cord was substantially reduced in RTX-treated rats. Although the proportion of μ opioid receptors on TRPV1- and non-TRPV1-sensory neurons may be altered by RTX treatment, it is less likely that the potentiated effect of spinal μ opioid agonists in RTX-treated rats is due to increased binding affinity of the μ opioid agonists or increased μ opioid receptor coupling to G proteins.
The mechanisms underlying this puzzling RTX-induced paradoxical change in opioid potency and μ opioid receptors in the spinal cord are currently unknown. One of the possibilities is that more functional μ opioid receptors may be distributed in non-TRPV1- than TRPV1-expressing afferent neurons. DRG neurons subserving specific sensory modalities are supported by different neurotrophins (Snider and McMahon 1998). In adulthood, approximately one-half of nociceptors are dependent on nerve growth factor (NGF) for trophic support; these neurons express the TrkA receptor for NGF and contain neuropeptides such as substance P and calcitonin-gene related peptide (Averill et al. 1995; McMahon et al. 1994; Molliver et al. 1995). The other half are sensitive to glial-cell-line-derived neurotrophic factor (GDNF); most of these DRG neurons possess receptors for GDNF, express TRPV1, and bind to IB4 (Bennett et al. 1998; Guo et al. 1999; Wang et al. 1994). DRG neurons expressing NGF receptors (mostly IB4- and TRPV1-negative) are important for normal pain perception. In this regard, rats and mice deprived of NGF during embryonic development by antibodies or gene targeting are unable to respond to painful stimuli (Crowley et al. 1994; Johnson et al. 1980; Smeyne et al. 1994). Furthermore, humans with mutations of NGF TrkA receptors or NGF beta gene are unable to detect pain (Einarsdottir et al. 2004; Mardy et al. 2001). Little is known how analgesics such as opioids modulate nociception transmitted by different phenotypes of primary afferent neurons. We have shown that the density of μ opioid receptors is ∼18% higher in IB4-negative than in IB4-positive (mostly expressing TRPV1) DRG neurons (Wu et al. 2004). Also the μ opioid agonists produce a greater inhibition of voltage-gated Ca2+ channels, especially the N- and P/Q-type, in IB4-negative than in IB4-positive DRG neurons (Wu et al. 2004). The present data, combined with the prior cellular study, lead to the prediction that the density of μ opioid receptors in TRPV1-negative sensory neurons may increase after RTX treatment.
Another possibility for this paradoxical finding is that RTX may alter the spinal cord circuitry so that the synaptic connection/interaction is reorganized somehow to facilitate the inhibitory effect of μ opioid agonists. We have demonstrated a small degree of sprouting of myelinated afferent terminals in the spinal lamina II after RTX treatment (Pan et al. 2003). It has been shown that morphine increases the activity of lamina II neurons but suppresses the firing of deeper dorsal horn neurons (Chen et al. 2005; Sastry and Goh 1983; Woolf and Fitzgerald 1981). Furthermore, the inhibitory action of morphine on deep dorsal horn projection neurons depends on its effect on GABAergic and glycinergic inputs to lamina II neurons (Chen et al. 2005). In RTX-treated rats, C fiber termination in the lamina II is largely removed, and sprouting of TRPV1-negative primary afferent terminals could re-target different lamina II neurons (Pan et al. 2003). This reorganization of the spinal dorsal horn circuitry may constitute a neuroanatomic plasticity for the potent inhibition of μ opioid agonists of nociceptive inputs transmitted through non-TRPV1 afferent neurons. It would be interesting to examine if TRPV1 and non-TRPV1 afferent terminals form synapses with different types of inhibitory and excitatory interneurons in the spinal dorsal horn after RTX treatment. Additionally, the absence of TRPV1-expressing afferent fibers may result in less nociceptive input to spinal dorsal horn neurons, resulting in enhanced response to μ opioid agonists. Hence further studies are warranted to determine how reorganization of the spinal dorsal horn circuitry after RTX treatment leads to increased analgesic effect of μ opioid agonists.
The potential differences in biochemical pathways responsible for the termination (receptor desensitization, internalization, and sequestration) of the μ opioid actions between TRPV1 and non-TRPV1 nociceptive afferent pathways should also be considered. The μ opioid receptor can be desensitized after activation by μ opioid agonists through phosphorylation by G-protein-coupled receptor kinases (GRKs) (Schulz et al. 2002). Phosphorylated μ opioid receptors are then bound by arrestins (Bohn et al. 1999; Whistler and von Zastrow 1998), which prevent further stimulation of G proteins and downstream signaling pathways by μ opioid agonists. The μ opioid receptor also can be phosphorylated by protein kinase C (Mestek et al. 1995; Narita et al. 2001). Differential expression of these signaling molecules in TRPV1- and non-TRPV1 primary afferent neurons may substantially influence the efficacy of μ opioid agonists. For instance, it has been shown that protein kinase C is preferentially expressed in IB4-positive (mostly TRPV1-expressing) DRG neurons (Molliver et al. 1995), and activation of protein kinase C reduces the inhibitory effect of μ opioid agonists on voltage-gated Ca2+ channels (King et al. 1999). This difference may contribute to a greater effect of μ opioids on voltage-gated Ca2+ channels in IB4-negative than in IB4-positive DRG neurons (Wu et al. 2004). Additional studies are necessary to determine if opioid desensitization and tolerance are altered in RTX-treated animals.
Although TRPV1 is well known for its critical role in thermal nociception, pain elicited by mechanical stimuli is far more clinically important. Thermal and mechanical nociception may be mediated by different (and overlapping to certain degree) subpopulations of distinct phenotypic nociceptors. In RTX-treated rats, mechano-nociception remains largely intact (Pan et al. 2003; Xu et al. 1997). Thus non-TRPV1 primary afferents appear important in the transduction and transmission of mechano-nociception. Nevertheless the phenotypes of primary afferent neurons specifically involved in mechano-nociception have not been identified. RTX-treated rats can be used to study the physiological function and pharmacological modulation of mechano-nociception transmitted by non-TRPV1 primary afferent neurons. Further studies on the differences in the opioid actions and opioid receptor desensitization between different phenotypic nociceptive afferent neurons will provide additional new information for the differential opioid modulation of modality-specific nociception. This new information will be important for a better understanding of the analgesic mechanisms of μ opioid agonists and provides a rationale for improved opioid therapies to treat different painful conditions.
This work was supported by National Institutes of Health Grants GM-64830 and NS-45602.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 by the American Physiological Society


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When Hillary Clinton used her Martin Luther King Day tribute to accuse Republicans of running Washington like an Old South "plantation" - she knew whereof she spoke.
In fact, when Hillary and Bill ran Araksnas, Dr. King didn't even have a holiday in his honor - at least not all to himself.
Instead, Arkansans celebrated a combination holiday that honored both King and Robert E. Lee, the Confederate general who fought to allow the South to keep blacks enslaved.
And that wasn't the only example of the kind of plantation politics that characterized the Clintons' rule in Arkansas. According to a 1997 Washington Times report:
"As governor of Arkansas, Mr. Clinton signed a law in 1987 that says the top blue star in the state flag symbolizes the Confederacy. Then-Gov. Clinton also issued proclamations designating a birthday memorial for Jefferson Davis, the president of the Confederacy.
"In addition, during his 12 years as governor, Mr. Clinton made no effort to overturn a state law that sets aside the Saturday before Easter as Confederate Flag Day."
In fact, life was so tough for African Americans on Bill and Hillary's Arkansas plantation that the NAACP sued Mr. Clinton under the federal Voting Rights Act of 1965.
"Plaintiffs offered plenty of proof of monolithic voting along racial lines, intimidation of black voters and candidates and other official acts that made voting harder for blacks," the Arkansas Gazette reported December 6, 1989.
The paper added: "the evidence at the trial was indeed overwhelming that the Voting Rights Act had been violated."
A three-judge federal panel ordered Gov. Clinton, along with Arkansas's then-Attorney General Steve Clark and then-Secretary of State William J. McCuen, to redraw electoral districts to maximize black voting strength.

subset: commoncrawl


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Pelosi Statement on FCC Measure for Greater Transparency in Politics
Washington, D.C. – Democratic Leader Nancy Pelosi released the following statement today after House Republicans on the Appropriations Committee reversed their position and will no longer attempt to block a Federal Communications Commission rule to require television broadcasters to post their political ad files online:
"Today, House Republicans reversed their opposition to a step for greater transparency in our politics. For a brief moment, Republicans saw the light, and as a result, America's voters will benefit from increased accountability, expanded disclosure, and more access to information about the campaign ads flooding our airwaves.
"Democrats have fought to ensure our campaigns are fairer, our politics is more open, and our elections are conducted on a level playing field. Earlier this year, the FCC's new rule marked a step forward in this effort, part of our broader drive to create a new politics free from special interests.
"With this small victory, we must keep moving ahead: to promote disclosure; to amend the Constitution and reverse the devastating effects of Supreme Court decisions that put special interests ahead of the public interest; to reform the system and empower the grassroots. We must ensure that the voices of the people determine the outcome of our elections, not the checkbooks of the few. We must build on today's shift by House Republicans and work together toward a future of openness in our politics.'


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The Rev. Wayne Murray says he is perplexed regarding the amount of money City Council awarded Hope on the Hill on Tuesday.
Hope on the Hill, a local youth-serving organization, is renovating the old Barr Street School and had asked for $500,000 from the city's hospitality tax grant fund.
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Northern Michigan woman designs for Olympic gold
Tue, 21 Jan 2014 01:03:05 GMT —
All eyes will be on the athletes at the upcoming winter Olympics, and one northern Michigan woman has a big part in how the figure skaters look under the bright lights.
Stephanie Miller has been designing costumes for both men and women skaters for more than 20 years.
â??I take stuff with me on vacation believe it or not because I can't be gone,â?? said designer Stephanie Miller.
Stephanie started figure skating in high school and quickly started making her own competition dresses.
â??I never dreamed in a million years that I would be doing stuff for Olympic skaters.â??
Setting up shop in her Kingsley home, Stephanie makes outfits for all levels of figure skaters. Her business got a boost when Meryl Davis and Charlie White won a silver medal while wearing her creations in Vancouver.
â??(People) just kept calling, and calling so sometimes we have to turn them away because we have too many dresses and we don't rush anything. We try to make it a custom made dress.â??
Stephanie designs the costumes, getting ideas from the Golden Globes, red carpet events and movies. From there her business partner puts the costume together, and Stephanie takes care of the rest, a product seen by millions of people across the world.
â??Pretty cool at first. Then everybody is calling you 'hey I saw your dressâ?|' now I see it a lot which it's still very exciting. I'm very humbled to the fact that here I am doing something like that.â??
The process takes about two months from start to finish before the costumes hit the ice.
â??I take great pride. I have thousands of pictures of every dress I've ever done from the lowest little skater to the top Olympian. I have pictures of all my stuff and I keep it proudly in albums upstairs.â??


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Nebraska's Best:
At their best.
Nebraska FFA State Convention
Nebraska FFA members and chapters will be honored for their accomplishments at the 89th Nebraska FFA State Convention held April 5-7, 2017. The convention will be at Pinnacle Bank Arena, the hotels in Lincoln's Haymarket, and the University of Nebraska-Lincoln East Campus.
FFA members, advisors and guests can support chapters and members receiving awards and listen to motivational speakers and state officer retiring addresses during convention sessions; be engaged in leadership workshops; watch students compete in Leadership Skills Events and attend the annual Career Fair. Students also participate in the state Career Development Events held on UNL's East Campus.
Convention Results
2017 Nebraska FFA Convention Results
The 89th Nebraska FFA Convention was held April 5, 6 & 7, 2017
2016 Nebraska FFA State Convention Results
2015 State FFA Convention Results
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The old saying that timing is everything is just as true in the world of sports as it is everywhere else, but the competitive nature of this realm means that good timing isn't enough to ensure success.
A perfect illustration of this can be found in the case of Bum Phillips' former Houston Oilers teams. This group certainly had good timing, as a trade in 1978 put Hall of Fame power running back extraordinaire Earl Campbell on the roster. Phillips had an offensive system that was tailor-made for a back like Campbell.
The ideal combination of coach and bell cow runner was a key reason that that from 1977 to 1980, the Luv Ya Blue crew was one of only six NFL teams to win at least 40 games (Dallas, Pittsburgh, Los Angeles, Denver and Oakland being the others).
This run of success led to a Super Bowl berth or win for all the other teams on that list -- but Houston couldn't manage even a division title in those years in large part because it had to battle Pittsburgh for that honor. That didn't stop the Oilers from reaching the playoffs and having postseason success, but they also ran into their black-and-gold division brethren in two AFC title games and lost both. Had this club come around at any other time, it probably would have made its own Super Bowl run, but it had the bad fortune of running into one of the best teams of all time.
There is a parallel between that Oilers situation and the current circumstances surrounding Michigan State Spartans quarterback Kirk Cousins. The junior is recognized as being a pretty good field general, but a closer look at the numbers shows he is much better than that.
Let's start with some of the basics. Cousins posted a 153.51 passer rating this season, a total that ranked 19th-best among FBS quarterbacks. He was tied for 20th in passing yards per attempt (8.5) and ranked ninth in completion percentage (67.5 percent). He also ranked 10th in the FBS in passer rating in the second half or overtime of games (172.95).


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Apr 13, 2004 | 320 Pages
"Powerful. . . . A challenging, disturbing portrait of a democratic hero, and an equally challenging case study of the democratic system." —The New York Times
"Rich in insight into Jackson's personality. . . . Burstein makes fair on his promise to look dispassionately at this most passionate of presidents. . . . A very readable, insightful analysis into the character and evolution of the American republic." —Plain Dealer
"Excellent. . . . A must-read for anyone interested in the presidency or early American history." –Flint Journal
"A useful, persuasively critical account of the development of Jackson's self-image as an honorable patriarch and champion of righteous government.." —The Washington Post Book World
"Impressive. . . . Persuasive. . . . Argues that the times shaped Jackson and thrust him into the White House as the first 'commoner' elected president because he so personified the young nation's bold, brash spirit and sense of destiny." –The Baltimore Sun
"In his ably drawn portrait…[Burstein] studies Jackson from many angles: as the orphan of the American Revolution, the self-taught orator, … and as the lanky husband who loved his stocky wife, Rachel, touchingly and fiercely." –The New York Times Book Review
"Well-researched and well-written.. . . Burstein, with his longstanding interest in the American mind, wants to show how we pick our national heroes." –Chicago Tribune


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The United Nations has played a pivotal role in addressing sustainable development ever since it defined the term in the Brundtland Report back in 1987. We should give them credit: the UN has been good at engaging all sectors of society, including business, on sustainability. That more companies continue to sign up for the UN Global Compact standards for responsible behavior speaks to the UN's ability to reach the private sector.
We're going to help the UN take that engagement one step further.
The Sustainable Development Solutions Network (SDSN), with 12 Thematic Groups, mobilizes experts from academia, civil society and the private sector to drive sustainable-development problem-solving at local, national and global scales.
The WBCSD has an important role in the SDSN. I'm serving as co-chair of the Role of Business Thematic Group with Klaus Leisinger, Chair of the Novartis Foundation for Sustainable Development. Our charge is to identify common solutions, highlight best practices and provide insights in the role of Business, to inform the high-level UN panel that is developing the Sustainable Development Goals – the successor to the Millennium Development Goals process which will end in 2015.
In January, we released a background paper on our priorities and our three-year work agenda: "Business: The key challenges to 2030/2050." In it, we've mapped out the challenges that must be solved quickly and outlined the action agenda for the Thematic Group. We know sustainability must be integrated into core business operations and strategies. And we know that externalities such as ecosystem services must be built into prices. What we need to do is make clear the actionable next steps that businesses and policy-makers must take.
Business, as the provider of innovation, management skills and financial resources, will play a role in almost all of the 12 Thematic Groups. So we'll also be consulting across all the Working Groups.
The SDSN also dovetails with the WBCSD's own work, as we transform Vision 2050 into a framework for action that we expect will drive companies to implement business solutions, at a broad enough scale to make a difference.
Business acts boldly and successfully when there is certainty, data and timelines. So we need to provide quantifiable, actionable and time-bound solutions for businesses in order to drive action. And that's what we'll do, through our involvement with the UN SDSN and through WBCSD's own work programs. That's my promise to you.


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Helloooo. i i just found out about something called Club Nintendo, when i was going through my games. And so you enter these codes on the site from your games and you can get prizes. there is this awesome Mushroom tote bag that i want for 250 coins, Sadly, after entering all my codes i only total up to 160. does anyone have any they don't need? o:
If so you can pm them to mee. D
Last edited by Pob3008; 03-21-2012 at 12:02 AM.


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This kind of doubles as an off topic thread for the web dev section when no one posts anything interesting they're working on. I don't think it's a bad thing. I'll post a redesign I'm working on once I'm on my laptop again.
This kind of doubles as an off topic thread for the web dev section when no one posts anything interesting they're working on. I don't think it's a bad thing. I'll post a redesign I'm working on once I'm on my laptop again.
Jagex is working on making RuneScape in html5 apparently
java left, html5 right
actually im reading further into it and this post makes me feel like an idiot.
ITT: Mostly 18 year olds and younger who've never have a real job and don't understand how finances in the real world work. Housing, utilities, and food all cost money.
Those of us who are older than two decades are sitting here loling at your misunderstanding of the world.
So, my company said they've had a good experience with me and want me to sign a new contract to collaborate with them during school which starts in two weeks. I'd be working from home for something like 12-14 hours a week with a laptop they give me.
They currently pay me 4.16 an hour which is pretty low but pretty much "standard wage" here in Italy. I was planning to ask for at least twice as that, or more, in order to sign with them.
What do you think?
you're an amazing designer tera, don't settle for minimum wage!
Well, I don't work as a designer there, I'm a developer. Their design offices are in the US.
Any chance of a set price per project? Might make things better for you.
Speaking of jobs.. Tbh I've never really considered working for anyone else career wise since about the age of 12, and I don't think I ever will. Even if I was a debilitated tramp I'd still have some kind of venture going on.
It'd really make me cringe working for a web development firm and getting paid a set wage for completing full designs, only to see the company was pulling in tens of thousands per project.
If you guys have any cool ideas for stuff that need investment, I'd probably be interested in grabbing a small share and possibly contributing design/ development work.. I'm pretty new to VC so the amount I could offer would be fairly low and it'll be really select, but nevertheless feel free to PM me and we can work something out.
You see SCop, working in a web dev company assures that you DO get a wage. Working as a free lancer does not guarantees that you will be paid, sure, you can earn much more, however, what if you don't get lucky that month and find no work? You're pretty much fucked.
And this also applies for, let's called them start-ups, as in, first of all you must have some capital beforehand, because you will not earn money for at least some time. And another problem - if your idea fails, you're also pretty much fucked.
And that's why I think a set-wage job is important. Really, once you have such a job, I believe you should start thinking about your own ideas as side projects and when they're mature enough, leave your current work and dedicate yourself to the project.
I know of a few freelance graphic designers, and their personal approaches by observing how they work.
One barely gets jobs and seems to spend all his time on Facebook going on about how he doesn't work for free.. even making beautiful images explaining how little he makes and how much he should be making. He uploads all his work onto Facebook and has no real way for people to contact him, aside from facebook.
His 'target market' seems to be small independent shops and bands, and people who want tattoos. All of these avenues have very little in the way of capital with which to pay him, so he shouldn't be expecting much more than 100 pounds per design, nor should he be pissed off when people ask him if he'll work for free.
Another spends time making a sharp, crisp, awesome corporate logo, and names his company after his own name (pretty simple right? Barely any creativity required for that but it works). He draws some select pieces of work that are of interest to his target market (cars designs) and puts them up on a nice portfolio website with a simple and clean theme.
He goes straight to every car and car design firm he can find, along with any companies tied to car design in some way, via email, to offer his services and price, and sends them a link to his portfolio. Within a day or two he lands a big contract and gets tens of thousands of pounds worth of work very quickly, which pays the bills for several months.
Another, who has been established for several years, offers his design services to companies like BMW and Audi to name a few.. He's somehow got away with charging them ridiculous amounts of money per concept he displays to them, and even if things aren't ever used, he still lands like the region of eight thousand pounds per iteration of design (half-weekly presentations or whatever) for some of his work. Of course he had to get to this stage first, which meant he did work for smaller companies and made less than he does now at some point in the past.
You have to remember that some huge companies probably hire ten different freelancers for the same job and just pick from the best one. When you've got millions coming in the door every day, having £100k to blow per week on some design projects is petty cash.
So yeah that's it. The 'trick' as a freelancer (can be applied to business as well, really) is to create work similar to what a customer already has, which will appeal to them. Stuff that matches their brand focus, if you will, and then contact them. Don't sit back waiting for jobs to come to you, or you'll end up behind a till at McDonald's, wondering why you aren't getting the recognition you deserve.
As for start-ups, it's a whole new ball game. If you can do stuff yourself the cost can be pretty minimal. Hosting for £5 a month and domains for £5 a pop (yearly). Where the ideas come from? Who knows. Experience as much as you can every day (even outside of the internet, although this is not essential) until inspiration strikes I suppose.
I never perceived working as a freelancer my business. I did it (and will do, probably) because I wanted to gain experience in the field and at the same time earn some money.
If you see yourself as a business then I do not think you can call yourself a freelancer.
The two aren't mutually exclusive.
Finished the blog redesign :) (live@ http://blog.rndm.de/)
If you got any problems feel free to tell me.
I want to add a shoutbox to my site. Any recommendations which one to use?
Also, are shoutboxes a good idea? Or is there something with similar functionality that works better.
I know what you mean. Not to mention most of them would degrade my site to a teenage girl's blog.
I don't really NEED a shoutbox but here's my situation.
It's a gaming website, we organize tournaments for League of Legends. I want a place where people can ask stuff, or generally talk and I can reply to them when ever I can. Something like comments on videos. I would use a forum but I think it pushes people away because they need to register.
Since it's a small website I don't see a problem with posting stuff as guests because I can remove it manually so a forum is an overkill.
Well, I have never worked with databases and I have no idea how I'd make it auto refresh.
Besides, I don't want to make it myself.
I don't need to communicate with the people I know. I want to be able to communicate with the visitors of my site.
You need some kind of registration system, you can see only the messages that are sent after you log in and it's a bit too big for what I want.
You obviously wouldn't host your own IRC server.
Either narrow the text, or go for two columns.
I made a new skin for the wiki I am an administrator of. Comments?
The CSS code I edited may be found here if anyone wants to check that out. Note that I have only limited CSS skill.


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The Cat in the Hat Visits Pierre Bossier Mall for Read Across Louisiana Day
Bossier City kids headed out to Pierre Bossier Mall over the weekend to celebrate Dr. Seuss' birthday at Read Across Louisiana.
Courtney Feliciano with the Bossier Association of Educators, who's also the curriculum instructor at Waller Elementary, tells KEEL News there were lots of special guests at the event.
"The Cat in the Hat is here," Feliciano says. "We've had Brandon Harris, who's the Parkway quarterback football star that's agreed to go play for LSU next year. We've had Representative Henry Burns, Representative Jeff Thompson." All those guests, and others, read popular Dr. Seuss books to the kids.
Bossier High School cheerleaders also handed out free books and cookies, as well as Cat in the Hat hats and Thing hair, all in an effort to encourage kids to read.
"We just can't stress enough how important reading is," Feliciano says. "And we want to help parents and kids get excited about reading and so that's why we gave out free books, to give the kids a chance to go home and enjoy their free book."
Check out photos from the event here:


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You are here
Keeping the farm in the family -- without farming
We are successful, third-generation rice growers with 3,500 acres of improved land. Until recently, all the owners worked on the farm, so ownership transition wasn't much of a problem. Now, some of our fourth generation are looking outside the farm for careers.
They all love the farm. All eight of them grew up here, but farming isn't in their blood like it is with my two cousins, my brother, and I. Recently we were talking about planning ownership in the future. The more we talked, the more depressed we got.
Three in the next generation want to farm, each from a different family. None of my children is interested. My partners thought I could just sell to them when I retire and it would be fixed.
But that got me thinking. I told them I had too much respect for what Granddad, my father, and my uncle did to build this farm to want to sell out.
I see a lot of potential for agriculture, especially rice here in the Delta, and I don't want my family to give up the future growth and profit.
But most important, I want my grandchildren to have an option to farm, something they'd lose if I sold. We're stuck. Do we have options?
Keeping a farm in the family over many generations is a dream shared by most families who own ag operations. But as W.Q. has found out, there's a gap between the dream and reality.
The challenge begins with the first heir who decides not to be a farmer. And the greater the number of heirs in a generation, the greater the probability that some will not want to farm. This brings up two hard truths:
- Forcing on- and off-farm heirs to be partners solely to keep the farm in the family has destroyed many families and almost as many farms.
- Expecting the farming heirs to buy out the defectors, generation after generation, drains precious capital out of a business that needs all it has.
These realities don't mean we have to give up the dream. But they have implications for planning to achieve it.
There really is no good reason to keep a multiowner family farm over the generations unless it meets two goals.
Goal 1: The farm must provide a superior return on investment in a form that is attractive to the owners and consistent with the family's values. This doesn't have to mean significant wealth growth. A family can accept average financial returns in favor of other values, as long as they're agreed on by all owners. This is where W.Q.'s generation must start their assessment and planning if they are going to move to on- and off-farm ownership.
Goal 2: The farm must positively promote on-going family unity. This can happen in many ways such as in a shared pride in family reputation, in regular meetings of owners (where defining the roles of off-farm owners is key), through creation and review of owner mission and vision, and with establishment of traditions like annual harvest weekends. But these will only happen if they are made to happen.
W.Q. does have options, but the interaction of these return/unity goals requires careful balancing. Family involvement must be part of the overall plan, yet separating management authority from long-term owner oversight is critical. A family farm is a fine goal, but it's far from easy to achieve.
We are successful, third-generation rice growers with 3,500 acres of improved land. Until recently, all the owners worked on the farm, so ownership transition wasn't much of a problem. Now, some of our fourth generation are looking outside the farm for careers.


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Hoist a mug for Kickstarter, once again giving another ambitious pirate the chance to make his salty dream a reality! Luke Ahearn's own dream is to influence younker to a life a piratin' and spellin', which seems a fine start to me. Check out his video, and if yer so moved, drop a coin or two!
A Pirate's ABC Adventure on Kickstarter
Comment (1)
Thanks for this mention! Means a lot.


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Home Design. Thursday , January 19th , 2017 - 14:02:49 PM
Quote from Private Home with Soft Wall Design :
The interior design of this cutting-edge house is applied in applied in spacious area in long term. The situation of the room is in minimalist attractive situation. They include the decoration for the hardwood floor with soft wall design. As in the living room, they apply the brown with red pattern and red stripes line on the black furry rug. There are also some other furniture included the light and dark brown sofa in one side. The furniture is charmed by appearing of the modern fireplace in short frame. Behind the living room, there is a dining table with its wooden furniture of the table and also the chairs.
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text: Okay. I got numpy to install okay, but now I'm having a problem with scipy. Now, the full output of the install command is really long by the time it hits the error, but I believe that these lines at the end specify what the error is: sh: line 1: f95: command not found sh: line 1: f95: command not found error: Command "f95 -fixed -O4 -target=native -c -c Lib/fftpack/dfftpack/dcosqb.f -o build/temp.macosx-10.3-fat-2.5/Lib/fftpack/dfftpack/dcosqb.o" failed with exit status 127 Can some one explain to me what this error means and how to go about correcting it? -- R. Padraic Springuel Teaching Assistant Department of Physics and Astronomy University of Maine Bennett 309 Office Hours: Wednesday 2-3pm


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Gold Traders Protect Your Longs
Gold traders should have exited today as prices are showing strong signs of lower prices in the near future. Gold slid over 4% this week so far, while gold stocks have dropped an average of 9%.
The weekly chart shows a very clear picture of resistance for gold which was reached last week and we are starting to see sellers step in now. Also my custom stochastic indicates gold has reached a short term top. Prices could retrace (pullback) to $700 level but I am hopeful that prices will hold around the $800 level. We will continue to watch it unfold.
Weekly Gold Trading Chart
Gold Stocks Lead the Price of Gold
In many cases gold stocks tend to lead gold. This daily chart below clearly shows a classic 5 wave counter trend rally and trend line breakdown. Gold stocks hit resistance last week and have been sliding lower since hitting their heads on the 200 moving average. Stochastic and MACD both signaled this is a high probability move, indicating there could be much more downside pressure.
Daily Gold Stock Chart
Gold Trading Fund and Analysis
The price of gold continues to have sellers stepping in this week. We continue to hold our core position as gold consolidates (pulls back) while short term traders take profits. We are looking for prices to drift lower and hold above the support trend line providing a reversal candle and pivot low with risk less than 3% for our next entry point. If gold stocks take another big hit this week then the HUI:GLD ratio will turn bearish and that's not good for the price of gold. All we can do now is to wait it out.
Daily GLD Exchange Traded Fund
The broad market as well as commodities have been holding their ground and making small gains for the past month. Fewer buyers seem to be at these higher prices, leaving me with a cautious vibe. Stick with your stops; you can always buy back in.
I will provide a full Special Report covering Gold, Silver and Oil this weekend. For more information please visit my website: Chris Vermeulen www.TheGoldAndOilGuy.com.


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New Wii owner here. Bought one off Craigslist awhile back. Great site! I've read a couple guides.
Only 2 questions (3 I guess...)
I am looking for some advice on what external HD to get?? Also, when I use an external HD does that HD become "wii only?"
Is there a HDD FAQ out there somewhere you can point me towards?
Hello and welcome to the wiihacks community.
I know you are anxious to get started; but, there are a few items to cover before you post your questions or start doing anything to your Wii.
Make sure you read the rules of this site. There are a number of tutorials and guides available to assist you. Many of our new members find the tutorials for softmodding any 3.1-4.1 Wii and any 4.2 Wii particularly useful. Make sure to either look through the site tutorials and guides or use the search function before asking questions. While the moderators, staff, and members of this community are willing to help you, some effort on your part to read, research, and follow the rules is expected. To get the best response to your question(s), post in the associated forum areas. This is very important as inappropriate posting could get you restricted to the reading room or even banned. If, after looking at the tutorials and trying to search, you still need help or just want to post a very basic type of question, go to the http://www.wiihacks.com/wiihacks-newbie-corner/.
Wiihacks also has a chat room on irc://irc.uicn/wiihacks. After reading the iRC rules, please feel free to join in the light-hearted chats and activities. While any iRC client can connect to #wiihacks on the UICN, ChatZilla, for those with Mozilla's Firefox browser, is easy to install and use.
If this site and our members have provided substantive help, you can Donate to the forum and get a nice banner under your name and gain access to the donator forums.
Here you are: http://www.wiihacks.com/recommended-...s-usb-hdd.html. Enjoy your stay with us.


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This is tough, this is going to be a long road.
How can I show her that I'm changing?
I have started IC and have done some reading, but I don't even really see her.
You did lie, and you did con. So, make up for that.
Want to change? Prove it to her.
Some reading? Do a buttload of reading. Even if you don't see her. It will make you a better person.
What can I do to prove it to her?
What are things you did?
If we did not have our son, she said she would have moved away (different city or state)
Well, don't mean to sound harsh here but . . .
Wayward is not the place for a BS to try to not sound harsh. Wayward is a place for BS's to not be harsh period. Please reread the forum description and the top post in this forum.
The people you do your life with shape the life you live
What does that look like? It's different for everyone, but if she needs space, give it to her, offer support when ever you can, respect her boundaries.
You have a son, what are you doing to support her and him? Even if she doesn't want to see you, are you still seeing your son?
Good luck, I hope you find your way to R.
Separated transitioning to D
The other thing of course, was his actions. He backs up what he says with what he does.
Unfortunately, with you guys living apart, this becomes much harder to show her. The good news is that she will still text with you, so at least you have some form of communication.
As for her heart being closed, I'm sure she feels that way now, but may not feel that way forever. So, try not to lose hope.


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Jayde came to the fiesta ready for some serious action. She showed her perky password at the door and access was granted by Jmac. He was mesmerized by her nice round ass as she walked inside. Jayde teased him by shaking it in front of his face and then by rubbing her pussy. When she was nice and wet, she asked him to fuck her hard. Jmac had to live up to his rep and he did. He pounded her until she had a few orgasms and then he unleashed the fiesta all over her face.


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Contact: Joanna Norris, Assistant DirectorDepartment of Media Relations and Events(904) 620-2102Morris Dees, trial lawyer and author, will discuss "With Justice for All" during his lecture at 7:30 p.m. on Monday, Oct. 17, at the University Center on the University of North Florida campus.Dees is the founder of the Southern Poverty Law Center, a nonprofit group he co-founded in 1971, which specializes in lawsuits involving civil rights violations, domestic terrorists and racially motivated crimes. Over the course of his career, he has done much to promote diversity. His efforts have resulted in many achievements, including the Civil Rights Memorial, lawsuits that bankrupted the Ku Klux Klan, imprisoned perpetrators of hate crimes and increased awareness of radical militias. Today, Dees focuses his attention on anti-government militias. He is the author of Gathering Storm: America's Militia Threat, A Lawyer's Journey, an autobiography, and Hate on Trial: The Case Against America's Most Dangerous Neo-Nazi. A graduate of the University of Alabama Law School, Dees has received numerous accolades in conjunction with his work at the Center. His honors include being named Trial Lawyer of the Year by the Trial Lawyers for Public Justice and receiving the National Education Association's Martin Luther King Jr. Memorial Award.This lecture is part of the Distinguished Voices Inquiry and Insight Lecture Series. Tickets for these free lectures can be ordered online at www.unf.edu. Click on the Fall 2005 Lectures link.-UNF-
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Twenty-year-old Camryn Bennett thought she knew exactly where her life was going. But after a wild night at the hottest club in downtown Raleigh, North Carolina, she shocks everyone - including herself - when she decides to leave the only life she's ever known and set out on her own. Grabbing her purse and her cell phone, Camryn boards a Greyhound bus ready to find herself. Instead, she finds Andrew Parrish.
Sexy and exciting, Andrew lives life like there is no tomorrow. He persuades Camryn to do things she never thought she would and shows her how to give in to her deepest, most forbidden desires. Soon he becomes the center of her daring new life, pulling love and lust and emotion out of her in ways she never imagined possible. But there is more to Andrew than Camryn realizes. Will his secret push them inseparably together - or destroy them forever?
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I never give reviews but WOW.....
I loved everything about this book! I had no idea what to expect when I started this book. I've been seeing great reviews on the Goodread website so I was excited to see it available thru Audible. The characters are beautiful, story is wonderful, and I fell head over heels fast with Andrew. He's hot, sexy, passionate, dominant, funny, strong, and yet he is sensitive. This book has some definitely steamy parts! I think this book was well written and well performed by Lauren Fortgang. This book hits you in all the right places! It has its sincere moments, funny moments, passionate moments and I was even a blubbering tissue blowing mess towards the end. This is by far is my new favorite book! It surpassed all of my other favorites straight to the top! BUY THIS BOOK!
I absolutely love Andrew Parrish. He is everything a girl could dream of for a perfect man!!
She did a fabulous job with all of them! I will definitely look for more of her narrations!
Yes Yes Yes..in fact, this book was worth being sleep deproved the next day for work!
I hope Audible gets the sequel that is due to come out in November. I wish Audible would start a genre on New Adult Romance not just Contemporary Romance. There are some very popluar books out that I'm dying to hear rather than just read. I would spend more money on credits if more new adult romance books were released on Audible!
- Jordy ♡♡♡♡
This one surprised me.
- Amazon Customer


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Hey! This thing is still a Work in Progress. Files, instructions, and other stuff might change!
TrayJet Printed Part Peeler
by mortinus, published
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Designed for quick / easy removal of printed parts from the makerbot print tray!
The tools that they (makerbot) provide are frankly not good enough. The squeegee thing just..doesn't work! I have more than once had to re-align my printer due to excess force required to break free the parts. This tool is modeled after a chisel or putty knife. The "hammer" is designed to give apply slight directly force exactly where it's needed.
Please print scaled 1.5x
I would recommend sanding the chisel tip
Added the chisel tool as stand-alone download. Please don't hold me responsible if you use your metal hammer and break anything!
100% Infill for everything.
.27 layer height
55 feed rate
110 heated bed temp
220 extruder head temp
AFter re-assembling the hammer/head multiple times it completely split the hammer down the middle. I will be updating the file to reduce the overall size of the join to allow for "tolerance stack" on the excess side of the printer (cause.)
Fixed Head Uploaded
Removed material from around thejoin cut-out which will prevent the hammer-handle from spreading the joint and splitting.
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Wedge: Ichiro's relatives appear safe in Japan
Wedge: Ichiro's relatives appear safe in Japan
Ichiro, starting his 11th season with the Mariners, declined to speak with reporters about the situation, saying, through interpreter Anthony Suzuki, he didn't have enough information at the time and had been unable to contact his family due to the power outages and phone difficulties.
But Mariners manager Eric Wedge said following Friday's 5-5 tie with the Indians that it appears Ichiro's relatives were safe.
"I just asked him and Anthony how they were doing and if everything was all right," Wedge said. "By all accounts in talking to them, they felt like the people they are close to are OK, at least the people they could get hold of. Obviously, with 4 million homes without electricity and everything else going on over there, it's just a horrible tragedy. It's a sad situation."
Ichiro, who grew up in Kobe, Japan, said earlier he has not been able to contact his family or ascertain their situation.
"We don't know yet, because cell phones and power are down. There are 4 million homes without power in Tokyo," Ichiro said through Suzuki, who is unrelated. "I have not got hold of my family yet."
Ichiro didn't travel with the team to its road game against the Indians in Goodyear, but only because Wedge had already decided to give many of his regulars the day off.
Suzuki said his own wife is currently traveling in Japan, but she is OK.
Hide Sueyoshi, the Mariners' director of Minor League and International Administration, is a native of Osaka, Japan.
"I've talked to my mother and friends in Tokyo," Sueyoshi said. "I tried to reach my friend in the Sendai area who works for the Rakuten Eagles [professional baseball team], but I could not reach him at all. The phone lines are just too busy, so I hope they are OK."
Sueyoshi said he went through a big earthquake while living in Osaka in 1995, while Ichiro was still in Kobe.
"I experienced that earthquake, so anytime it happens it's tough for me to see," Sueyoshi said. "I just hope all the people are OK. I know it's a lot of confusion."
Sueyoshi said the Eagles are in Spring Training in the area of the quake.
"They have started exhibition games though, so I don't know where they are," he said. "They were playing yesterday, but I could not reach them. The phones are just too busy."
Mariners CEO Howard Lincoln issued a statement on behalf of the club's ownership regarding the quake. The club is owned by Nintendo, the Japanese-based video game corporation that also has its U.S. headquarters in Redmond, Wash.
"The Mariners would like to join with people from around the world in extending our sympathy to the many families affected by the devastating earthquake and tsunami centered in Sendai," Lincoln said.
"The Mariners and the city of Seattle have a long and close relationship with the people of Japan. Particularly close to all of us here at the Mariners are the many Nintendo employees and their families in Kyoto, Tokyo and other parts of Japan and the many, many Mariners fans all across the country. Our thoughts and best wishes are with the families as they face this great challenge."
Greg Johns is a reporter for MLB.com. Follow him on Twitter @GregJohnsMLB as well as his Mariners Musings blog. This story was not subject to the approval of Major League Baseball or its clubs.


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After a season of Tyron Smith at right tackle and Doug Free at left, the Dallas Cowboys are open to the idea of flipping the two, employing their 2011 first round pick Smith as the protector of Tony Romo's blindside, in 2012.
In the introduction of the Cowboys' new-look coaching staff on Thursday, head coach Jason Garrett responded to the idea, which has been floating around on blogs and the like since season's end if not before then, by acknowledging that the team will consider it as they move into the offseason.
"It's certainly a possibility," Garrett said, per ESPN Dallas. "That was a possibility when we first drafted Tyron and one of the many things that was attractive about him was his physical ability to play both right and left tackle in this league. And if you remember, we gave him a chance a little bit early in training camp to have a couple of days playing the left side. He played two years at [Southern California] as a right tackle. He was a 20-year-old kid who didn't have an offseason, so we felt like, hey, we gave him a little snap shot of that, probably best for him right now to stay on the right side and he did a really good job for us this year.
"But he does have the physical traits as does Doug to play both sides. It's something we'll talk about as we continue to watch the tape."
Stay on top of the latest Dallas Cowboys news with NBCDFW.com. Follow NBC DFW on Facebook or have breaking Cowboys news sent directly to your phone with Blue Star SMS alerts. Text "bluestar" to 622339 (NBCDFW) to subscribe to this free service. Read more about it here.


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Category Archives: Reminder
Grub Embedding, blocklists and bios_grub partitions
This has tripped me up so many times, and the parted invocation needed to fix it is hard to find on google. The issue is that with GPT, grub won't have room to embed all of it's needed files, leading … Continue reading
Posted in Reminder Tagged diagnoses, gotcha, gpt, grub, partitioning Leave a comment
aptitude forbid-version
A quick one as a note to anyone who stumbles upon a counterintuitive part of aptitude that tripped me up- You can stop aptitude from every installing a version of a package with aptitude forbid-version package=version And the docs mention … Continue reading
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Mental note on Windows 7 under ESXi 4.0
For the last year or so I've been running windows 7 under ESXi and due to a few issues I've had to re-install more than once. Each time I go through the install process I seem to bump into a … Continue reading


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Asymptotic Properties of Residual Based Tests for Cointegration
This paper develops an asymptotic theory for residual based tests for cointegration. These tests involve procedures that are designed to detect the presence of a unit root in the residuals of (cointegrating) regressions among the levels of economic time series. Attention is given to the augmented Dickey-Fuller (ADF) test that is recommended by Engle-Granger (1987) and the Z(a) and Z(t) unit root tests recently proposed by Phillips (1987). Two new tests are also introduced, one of which is invariant to the normalization of the cointegrating regression. All of these tests are shown to be asymptotically similar and simple representations of their limiting distributions are given in terms of standard Brownian motion. The ADF and Z(t) tests are asymptotically equivalent. Power properties of the tests are also studied. The analysis shows that all the tests are consistent if suitably constructed but that the ADF and Z(t) tests have slower rates of divergence under cointegration than the other tests. This indicates that, at least in large samples, the Z(a) test should have superior power properties.
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|51 pages
|Date of creation:
|Date of revision:
|Jul 1988
|Publication status:
|Published in Econometrica (January 1990), 58(1): 165-193
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| Postal: Yale University, Box 208281, New Haven, CT 06520-8281 USA
Phone: (203) 432-3702
Fax: (203) 432-6167
Web page: http://cowles.yale.edu/
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| Postal: Cowles Foundation, Yale University, Box 208281, New Haven, CT 06520-8281 USA
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Simple and Robust Endpoint Management
CLI Device Manager is a clean user interface allowing IT managers to deploy, configure and maintain CLI thin client devices instantly. CLI Device Manager reduces ongoing support costs by easily managing and deploying device updates. The software client included in every CLI device provides an interface that simplifies the integration of CLI thin clients with industry-standard protocols including; SNMP, FTP and TCP/IP. With Support for Microsoft SQL database CLI Device Manager can support thousands of devices from a single console.
Device Manager Installation Tutorial
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The end of earnings season does not mean the end of excitement for us at Insider Monkey; we keep our ears to the ground for the round of 13F filings that usually post during and after each quarterly earnings season. Hedge funds are required by the SEC to disclose many of their equity, option, and bond positions, and we analyze these documents to glean the most impactful information out of them each quarter. Our research has led us to find that the most popular small cap stocks amongst hedge funds have outperformed the market by 18 percentage points per year (learn more about this phenomenon here). Billionaire Ken Griffin of Citadel Advisors, LLC recently made his filing public; read on to see our take on his newest picks during Q4 2012.
Citadel's quantitative investing model and $65bn in AUM enables the fund to trade and hold thousands of positions at a time. Out of the 790 new stocks purchased in the last quarter of 2012, ADT Corp (NYSE:ADT) was given the most capital, with 0.23% of portfolio devoted to the security company. ADT commands 25% of the market share of the $13bn industry in which it operates. The company reported earnings at the end of last month, delivering a 13% rise in net earnings year over year and announcing a $600m common stock buy back from Credit Suisse International. The stock is still trading at a 15% deficit to analyst's one year price targets. Doug Silverman of Senator Investment Group holds over $200mm of ADT Corp (NYSE:ADT) (view his other holdings here).
Gulfport Energy Corporation (NASDAQ:GPOR) was Citadel's second-largest new position. The independent oil and gas company has a relatively small market cap of $2bn and operates primarily in the Louisiana Gulf Coast and in the Permian Basin. GPOR announced a public offering of 7.75mm shares of common stock in mid-February; the dilution brought on a drop in share price, contributing to the -6.3% depreciation the stock has returned since the start of 2013. Despite the near-term fall, Wall Street has high hopes for GPOR and its future site production, with most analysts issuing a Buy rating for Gulfport Energy Corporation (NASDAQ:GPOR). Billionaire Jim Simons has a conservative $5.5mm invested (see his top picks here).
Autodesk, Inc. (NASDAQ:ADSK) is a strong player in the CAD (computer-aided design) industry with its comprehensive and knowledgeable business solutions. Despite missing every earnings announcement in 2012, ADSK managed to beat its latest announcement in February, which came on the heels of positive ratings acknowledgement from Wedbush, RBC Capital, and Goldman Sachs in January. Higher revenues and higher earnings versus the prior-year quarter set the precedent that ADSK would like to continue in 2013, as the company guided revenue growth for this fiscal year. Two Sigma Advisors beefed up its position substantially according to its most recent 13F (check out the fund's portfolio here).
Which other stocks found their way into Citadel's portfolio?
Cobalt International Energy, Inc. (NYSE:CIE) was another new oil and gas play for Citadel; contrary to GPOR, CIE's main exploration areas lie in the Gulf of Mexico and in offshore West Africa. The stock has suffered through the past twelve months, losing 22% of its value, with a significant drop occurring in January after the announcement of a 40mm share secondary public offering. Despite this, Wall Street is once again bullish on many of these smaller exploration companies, advocating accumulation and projecting much higher price targets for the future. Citadel initiated this position as a corporate bond purchase. Dallas-based HBK Investments made a similar debt investment in CIE (see the fund's top five positions here).
Textron Inc. (NYSE:TXT) was the fifth-largest new addition to Citadel's portfolio, comprising 0.07% of the fund's assets. Also a bond purchase, Griffin devoted $43mm towards the fixed income investment. Textron operates in a wide number of industries, including defense, finance, and aerospace (Cessna jets, Bell helicopters). The company's recent Q4 2012 earnings announcement in January showed a slight miss on the surface, although net earnings gave a profit and revenue grew over the same quarter from last year. Billionaire Steve Cohen joins Griffin in holding TXT bonds (view SAC Capital Advisor's full holdings here).
Disclosure: I do not own shares of any stocks mentioned in this article.