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Peripheral nerve injury leads to neuropathic pain, but the underlying mechanisms are not clear. The TRPV1 channel expressed by nociceptors is one receptor for noxious heat and inflammatory molecules. Lumbar 4 spinal nerve ligation (SNL) in mice induced persistent heat hyperalgesia 4–10 days following injury. The heat hypersensitivity was completely reversed by the TRPV1 antagonist A-425619. Furthermore, DRG neurons were isolated from the injured L4 ganglia or adjacent L3 ganglia 4–10 days after L4 SNL. Whole-cell patch clamp recordings were performed and heat stimuli (22–50°C/3 sec) were applied to the soma. Neurons were classified by soma size and isolectin-B4 (IB4) binding. Among directly injured L4 neurons, SNL increased the percentage of small-diameter IB4 positive neurons that were heat sensitive from 13% (naive controls) to 56% and conversely, decreased the proportion of small IB4-negative neurons that were heat sensitive from 66% (naive controls) to 34%. There was no change in IB4 binding in neurons from the injured ganglia. Surprisingly, in neurons from the adjacent L3 ganglia, SNL had no effect on the heat responsiveness of either IB4 positive or negative small neurons. Also, SNL had no effect on heat responses in medium-large diameter neurons from either the injured or adjacent ganglia.
Neuropathic pain results from damage or dysfunction of the peripheral or central nervous system. Common causes include carpal tunnel syndrome, nerve compression by neuromas, tumors or herniated disks, and surgical procedures.38, 39 Patients with neuropathic pain frequently experience spontaneous pain as well as stimulus-evoked pain including heat or cold hyperalgesia, or mechanical allodynia.
Neuropathic pain is thought to be driven by functional changes in directly injured fibers as well as changes in fibers that lie adjacent to the injured axons.7, 15, 34, 57, 64 The injured distal axons undergo degeneration which recruits inflammatory processes around the adjacent intact fibers.42, 55 Nociceptors are broadly categorized as either myelinated A-fibers that transmit noxious mechanical and heat stimuli, or unmyelinated C-fibers that respond to intense heat, cold and/or mechanical stimuli. C-fiber nociceptors are further classified into peptidergic and non-peptidergic, and can be differentiated by isolectin B4 (IB4) binding, as non-peptidergic neurons primarily bind IB4.3, 53 These two groups appear to provide input to two distinct pain pathways from the skin to the brain.5, 69 Little is known about nerve injury-induced functional changes in peptidergic and non-peptidergic/IB4-binding nociceptors that may contribute afferent input toward neuropathic pain.
The molecular basis for neuropathic pain symptoms is largely unknown. One critical transducer in nociceptors is the TRPV1 channel, which is activated or sensitized by natural and endogenous stimuli including noxious heat (>43°C), protons, N-arachidonoyl-dopamine (NADA), leukotriene B4, anandamide, and 12- and 15-(S) HPETE lipoxygenase metabolites.9, 26, 59, 68 Thus, besides activation by heat, TRPV1 may also contribute to injury-induced hypersensitivity via its activation or sensitization by endogenous ligands. The involvement of TRPV1 in inflammatory pain is well established,8, 12 but its role in neuropathic pain is controversial. In some animal models of neuropathic pain, mice lacking the TRPV1 channel develop the full magnitude of behavioral mechanical allodynia and heat hyperalgesia.8 However, other studies show that TRPV1 mRNA and protein levels are substantially upregulated in neurons that course adjacent to injured axons, with the greatest increase occurring in adjacent myelinated A fiber neurons.18, 25, 41, 49 Furthermore, TRPV1 antagonists effectively block nerve injury-induced mechanical allodynia in animal models.24, 48
Previous studies have assessed the contribution of TRPV1 to neuropathic pain via analysis of protein or mRNA levels and pain behavior assays, but no study has investigated injury-induced changes in TRPV1 function in sensory neurons. On a functional level, TRPV1 may collaborate in heterotetramers with other TRP channels,11, 23 or exist in multiple splice variants,62 and these configurations could be missed by protein or mRNA analyses. Therefore, we used patch clamp recordings to investigate nerve injury-induced changes in membrane TRPV1 function to natural heat stimuli.
Experiments were performed in adult male wild type mice (C57BL/6J) and TRPV1-null mice (B6.129S4-TRPV1tm1Jul; Jackson Laboratories, Bar Harbor, ME). Ages ranged between 10–16 weeks, and body weight ranged 20–30 g. All procedures were approved by the Medical College of Wisconsin Animal Care and Use Committee and were performed in accordance with the policies of the International Association for the Study of Pain and the National Institutes of Health.
Ligation and transection of the L4 spinal nerve was performed in mice with minor modifications to the widely-used Chung model for neuropathic pain in rats. 27 Adaptations were made to account for the neuroanatomical differences documented between rats and mice.50 Unlike rats where 98% of sciatic axons arise from the L4 and L5 spinal nerves,58 in mice, most fibers in the sciatic nerve arise from the L3 and L4 spinal nerves, with a smaller (< 20%) contribution from the L5 spinal nerve.50 Mice were randomly assigned to naive, SNL or sham groups. Under deep Avertin anesthesia, the paraspinal muscles (L3-S1 level) were bluntly dissected and the L5 transverse process was removed to expose the ventral ramus of the L3 and L4 spinal nerves. The L4 spinal nerve was tightly ligated with silk suture (6-0; Look, Surgical Specialties Corporation, Reading, PA) and transected distal to the ligation. In sham-operated mice, the L5 transverse process was exposed but not removed and the L4 spinal nerve was not touched. Naive, un-manipulated mice were included as controls to measure sham-surgery induced effects. The position of the ligature was examined post mortem by counting the dorsal root ganglia (DRG) embedded in the intervertebral foramen beginning with the DRG just caudal to the last rib as T13, the next caudal DRG as L1, etc. Neurons in the L4 DRG were considered directly injured and neurons in the L3 DRG were considered adjacent to the injury.
The radiant heat paw withdrawal test was used to assess heat hypersensitivity.6, 22 Mice were allowed to freely move on a glass floor surrounded by a plexiglass chamber. Radiant noxious heat was applied to the plantar hind paw. Ipsilateral and contralateral sides were tested in alternation and 4–5 measurements were obtained for each paw at each time point. Paw withdrawal latencies were assessed approximately 30 minutes after intraperitoneal injection of a selective TRPV1 antagonist, A-42561916 (Abbott Laboratories, Abbott Park, IL), or vehicle. The vehicle contained 34% weight by volume hydroxypropyl beta cyclodextrin in 10% sterile DMSO/dH20.
The L3 and L4 ganglia were removed from the operated side 4–10 days (median 7 days) after SNL or sham surgery. For naive controls, the L3 and L4 ganglia were removed bilaterally from a non-manipulated mouse and dissociated separately. Cells were isolated as previously described.13 Briefly, DRGs were incubated with collagenase followed by trypsin (Sigma-Aldrich, St. Louis, MO) and mechanically dissociated into single cells. Cells were plated onto poly-L-lysine coated coverslips. No exogenous growth factors were added. Recordings were performed 2–24 hr after plating.
Whole-cell patch-clamp recording was performed as described.13 Briefly, fire-polished glass electrodes (3–6 MΩ resistance) were filled with solution containing (in mM): KCl, 135; NaCl, 10; MgCl2, 1; EGTA, 1; NaGTP, 0.2; ATPNa2, 2.5; HEPES, 10; pH 7.2; osmolarity = 290 mOsm. The recording chamber was superfused with solution containing (in mM): NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 1; HEPES, 10; glucose, 10; pH 7.4; osmolarity = 310 mOsm. All recordings were performed at room temperature (23 ± 1°C). Soma size was estimated by a calibrated eyepiece reticle. Only neurons isolated from neighboring cells were used.
Membrane voltage or current was clamped using an EPC-9 amplifier run by Pulse software (version 8.78; HEKA Electronic, Lambrecht, Germany). Neurons were included if they formed a tight seal (> 1 gigaohm), had a stable resting membrane potential <−40 mV, and exhibited an action potential (AP) overshoot. Series resistance was compensated by > 60%. The presence of an inflection on the falling phase of the AP was determined by taking the first derivative (dV/dt) of the AP waveform as described13 and a late negative component indicated an inflection.
To measure whole-cell currents, membrane voltage was clamped at −70 mV. Capsaicin (1 μM; Sigma-Aldrich) was dissolved in 1-methyl-2-pyrrolidinone 13, 46 and applied for 10 s using a nonmetallic needle placed 50–100 μm from the soma.13 The vehicle had no effect on control, injured L4 or adjacent L3 neurons (data not shown). The criterion for a capsaicin response was an inward current ≥ 40 pA.6
Heat stimuli were applied as a fast ramp of heated buffer (22°C to 50°C in 3 sec) 50–80 μm from the soma using a custom-made heat stimulator designed by Dr. Ivan Dittert.14 The peak stimulus temperature was set at 50 °C because higher temperatures can induce irreversible damage to the membrane.36 The temperature was measured at the tip of the heat stimulator and the actual temperature at the soma membrane was approximately 1–2°C lower.
For pharmacological inhibition of TRPV1, neurons were first tested with a heat ramp in normal buffer. Following a 2 min superfusion with buffer containing the TRPV1 antagonist A-425619, a second heat stimulus was applied. After a 2 min washout with normal buffer, a third heat ramp was applied.
Immediately after recording, neurons were incubated with 10 μg/ml isolectin B4 (Bandeiraea Simplicifolia) conjugated to fluorescein isothiocyanate (IB4-FITC; Sigma-Aldrich) for 10 min and the IB4-FITC staining was visualized. A neuron was considered IB4 positive if it had a continuous green ring around the perimeter.13 Neurons with a soma diameter ≤ 26 μm were considered "small" and neurons >26 μm in diameter were considered "medium-large." The majority of IB4-positive neurons are ≤ 26 μm diameter and 95% of these IB4-positive neurons are negative for the 200 kD neurofilament antibody N52, a marker for myelinated neurons.13 Medium-large neurons were classified into those with- and without a somal AP inflection because a tight association between a somal AP inflection and nociceptive response properties has been described for myelinated neurons.17, 28, 51 For the naive and sham-surgery control groups, the response properties of neurons from the L3 and L4 ganglia were not different and thus, were combined.
DRG neurons were loaded with 2.5 μg/ml Fura-2 AM (Invitrogen, Eugene, OR) in extracellular buffer containing 2% BSA. Images of Fura-2-loaded neurons (excitation at 340 and 380 nm) were captured with a cooled LCD camera (CoolSNAP fx, Photometrics, Tucson, AZ; exposure time 2 sec). The ratio of fluorescence intensity (340 nm/380 nm) was analyzed using MetaFluor (version 126.96.36.199, Molecular Devices, Sunnyvale, CA). Background fluorescence was corrected by subtracting an image taken from a region of the coverslip devoid of cells or debris. The criterion for a response was ≥ 20% increase above the baseline ratio. A test solution of 50 mM K+ was used to depolarize neurons, increase intracellular Ca2+ and thereby, differentiate neurons from non-neuronal cells. After imaging, cells were incubated with IB4-FITC as above; IB4-FITC staining was visualized in neurons that responded to 50 mM K+.
Values are given as either mean ± SEM or median and quartile range. Differences in the current densities between different groups were compared using a Kruskal-Wallis test. Percentages of neurons responding to heat were compared using a Chi-square test followed by Fisher's exact test. For behavioral experiments, the overall main effect was assessed by either one-way ANOVA or two-way repeated measures ANOVA, followed by paired t-test comparisons at each time point.
First we performed an initial time course to determine the onset and extent of behavioral heat hypersensitivity induced by L4 spinal nerve ligation (SNL). By day 4 after SNL, mice developed prominent heat hypersensitivity in the paw ipsilateral to SNL compared to either the contralateral side or baseline before surgery. The heat hypersensitivity persisted through day 10 (Fig. 1A). In order to determine the contribution of the TRPV1 receptor to the heat hypersensitivity, we treated separate groups of SNL-injured mice with either the TRPV1 antagonist A-425619 (200 μmol/kg, i.p.) or vehicle 30 min before each behavioral test on days 4, 7 and 10. Vehicle-treated mice with SNL exhibited behavioral heat hyperalgesia over the 4–10 day time course that was similar to the initial L4 SNL group (Fig. 1B). In contrast, acute pharmacological inhibition of TRPV1 with A-425619 completely reversed the SNL-induced heat hypersensitivity at all time points (Fig. 1C and 1D). Heat responses in the contralateral paw were not affected by A-425619. These data indicate that acute pharmacological inhibition of TRPV1 completely reverses nerve injury-induced heat hyperalgesia.
Since the paw withdrawal test measures a reflex that includes DRG neurons as the sensory component, we next determined whether functional upregulation of TRPV1 in DRG neurons contributes to the nerve injury-induced heat hypersensitivity.
First, we assessed heat responses in neurons from naive mice. A subpopulation of neurons exhibited a robust inward current that rapidly returned to baseline immediately after the heat ramp (Fig. 2A). All other neurons exhibited a small non-specific leak current during the heat ramp (Fig. 2B). Thus, the criteria for an authentic heat response was a clear heat threshold evident by onset of a fast rising inward current as previously documented.36 Repeated heat-evoked currents exhibited little desensitization when applied at 2 min intervals (Fig. 2A).
A total of 32% (31/98) neurons of all sizes from naive mice responded to heat. Among small-diameter neurons, 51% (27/53; range: 16.3–25.3μm diameter) responded to heat compared to only 9% (4/45) of medium-large neurons (range: 26.9–47.3μm diameter). Heat activation thresholds in all naive neurons ranged from 42 to 47°C, with a mean threshold of 44.9 ± 0.2°C.
Further stratification of the heat-sensitive small neurons into IB4 positive and negative subgroups revealed that the large majority (92.6%; n=38) of heat-sensitive small neurons were IB4 negative, whereas only 7.4% (n=15) were IB4 positive. Hence, among all small neurons tested, significantly more IB4 negative neurons responded to heat (65.8%), compared to just 13.3% of IB4 positive neurons (Fig. 2C; P = 0.0007; Fisher's exact test). This differential heat-sensitivity between IB4 positive and negative small neurons paralleled the sensitivities the two subgroups to capsaicin (1 μM; Fig. 2D). Neither the average magnitude of the heat current (IB4−: 141 ± 33.8 pA/pF; IB4+: 208.6 pA/pF) nor the heat thresholds (IB4−: 45.11 ± 0.5°C; IB4+: 44.5°C) were different between the two groups. The low percentage of IB4 positive neurons responding to heat in this study (13.3%) is different than our previous report where 49% responded to heat.54 This difference is likely due to the shorter time in culture (on average less than 12 hr in the current study versus 27 hr in the previous study) and possibly to a shorter duration heat ramp (3 sec in current study versus 10 sec in previous study). The low heat-sensitivity of medium-large neurons corresponded with their low capsaicin sensitivity (Fig. 2E).
Since DRG neurons are known to express heat sensitive channels other than TRPV1,10, 19, 33, 53 we investigated the proportion of heat-evoked responses due to activation of TRPV1 channels by using both acute pharmacological inhibition of TRPV1 and genetic ablation of TRPV1. The TRPV1 antagonist A-425619 (1μM) completely and reversibly inhibited heat-evoked currents in small diameter neurons (Fig. 3A, B), leaving only a small, residual non-specific current. Second, none of the small-diameter DRG neurons isolated from TRPV1 −/− mice (n=29) responded to heat, compared to 51% of small neurons from C57BL6 wild type mice (p < 0.0001; Fisher's exact test). In medium-large neurons, all heat currents had kinetics identical with the heat currents from small neurons and the percentages of heat- and capsaicin-responsive neurons were the same. Together with our observation that the heat thresholds in naive neurons are characteristic of TRPV1,9 our findings indicate that at the level of the cell soma, all heat-evoked currents in naive DRG neurons are mediated via activation of TRPV1.
In mice, the sciatic nerve receives the majority of its axons from the L3 and L4 spinal nerves, and a small contribution from the L5 nerve.50 Therefore, after L4 SNL, the L4 DRG contains all directly injured neurons whereas the L3 DRG contains somata that have intact axons, and some of these axons course adjacent to injured axons. Since pain following nerve injury involves damage to nerve fibers, degeneration of the injured nerve fibers, and local inflammation, 61 we compared directly injured and adjacent neurons to naive neurons. Because sham surgery involves local inflammation caused by the skin and muscle dissection, we also compared neurons from sham operated mice to SNL mice to identify the specific contributions of the nerve injury component.
Spinal nerve ligation affected the heat responsiveness of small neurons from the injured L4 ganglia. The percentage of IB4 positive neurons that were heat responsive increased by 4-fold, from 13% in naive controls to 56% in injured L4 neurons (Fig 4A). In contrast, SNL decreased the percentage of IB4 negative neurons that were heat sensitive from 66% in naive controls to 34% in injured L4 neurons (Fig. 4B). However, SNL had no effect on the heat-responsiveness of either IB4 positive or negative small neurons from the adjacent L3 ganglia (Fig. 4A, B). Sham surgery had no effect on the heat responsiveness of either population of small neurons. The average amplitude of the heat-evoked currents did not change after injury in any group (Kruskal-Wallis test, Fig. 4C, D).
To determine whether the injury-induced increase in heat-responsive IB4 positive neurons was due to TRPV1 or another heat-sensitive channel, we used the TRPV1 antagonist A-425619 (1μM). Treatment with A-425619 completely and reversibly blocked all heat-evoked currents in IB4 positive L4 neurons after SNL (n=5), IB4 negative L4 neurons after SNL (n= 8), and all adjacent L3 neurons after SNL (n=2 IB4 positive; n =10 IB4 negative). Furthermore, A-425619 blocked all heat responses in sham controls (n=4 IB4 positive; n=16 IB4 negative). Thus, all heat-evoked currents after SNL were mediated entirely by TRPV1.
One explanation for the divergent changes in heat responsiveness of IB4 positive versus negative neurons after axotomy (L4 SNL group) may be a change in the IB4 staining. For example, some former IB4 negative neurons may have gained IB4 staining after injury, thereby increasing the apparent heat responsiveness of IB4 positive neurons. Thus, we quantified IB4 staining in small-diameter L3 and L4 DRG neurons from naive mice, or from mice 7 days after SNL. To verify that all cells examined were viable neurons, cells were tested by depolarization with 50 mM K+ using Fura-2 calcium imaging, prior to staining with IB4-FITC. Cells that responded with an increase in Fura-2 ratio > 20% over baseline during exposure to 50 mM K+ were considered healthy neurons. In contrast to findings in rat, 21, 32 the frequency of IB4 binding among small neurons did not change in injured L4 neurons compared to naive controls in mouse (Fig. 4E). This suggests that the increase in heat-responsive IB4 positive injured neurons and decrease in heat-responsive IB4 negative neurons was not due to changes in IB4 binding. However, IB4 binding decreased in neurons adjacent to injury from 54% in naive L3 neurons to 30% in adjacent L3 neurons after L4 SNL (p<0.0001, Fisher's exact test).
Among medium-large diameter neurons, SNL had no significant effect on heat response in either the injured L4 neurons or adjacent L3 neurons compared to naive neurons (Fig. 5A). However, injured L4 neurons were significantly less responsive to heat compared to sham controls (Fig. 5A). The difference between sham neurons and injured L4 SNL neurons may be because sham neurons are exposed to some inflammation from surgery whereas the L4 SNL neurons are exposed to axotomy and inflammation.
One criterion to identify putative nociceptors among the medium-large somata after isolation is the presence of an inflection on the falling phase of the action potential.17, 28, 51 All four heat-sensitive neurons in the medium-large naive group exhibited a prominent inflection on the somal action potential. After L4 SNL, significantly more L3 adjacent neurons expressed an inflection compared to either naive or sham neurons (Fig. 5B). This finding agrees with changes reported by others37 and indicates that the presence of an inflection on the somal action potential is not a stable criterion for defining myelinated nociceptors in injury models.
Here we show that peripheral nerve injury in mice differentially regulates the heat response properties of directly-injured small neurons during the same time course of behavioral hypersensitivity to noxious heat. Furthermore, TRPV1 inhibition completely blocks the heat hypersensitivity at both the sensory neuron membrane and the behavioral levels, indicating that in mice, TRPV1 mediates the heat hyperalgesia induced by spinal nerve ligation.
Whereas IB4 positive neurons from non-injured mice rarely respond to noxious heat, spinal nerve ligation increased their heat responsiveness 4-fold, such that after injury over 50% of the injured IB4 positive neurons responded to noxious heat. In contrast, IB4 negative neurons which are normally very responsive to noxious heat, were decreased in heat sensitivity by spinal nerve ligation. These differential effects of injury on functionally distinct subpopulations of small neurons were not likely due to underlying changes in IB4 binding because the percentage of neurons binding IB4 after injury was unchanged in mice. Other studies in rats21, 32 have shown a marked decrease in IB4 staining in DRG neurons after injury. Glial cell line-derived neurotrophic factor (GDNF) is critical for the survival and function of IB4 positive neurons.2, 43, 67 The differential effect of injury on IB4 binding in mice verses rats may be because mice and rats express different profiles for GDNF production by Schwann cells and satellite cells after nerve injury. 20, 45
Three pieces of evidence indicate that the heat-evoked current in the somata of all neurons from either naive mice or injured mice is mediated entirely by TRPV1. First, a selective TRPV1 antagonist, A-425619, completely and reversibly inhibited all heat-evoked inward currents in naive, sham, SNL injured and SNL adjacent neurons. Second, none of the neurons from TRPV1-mutant mice responded to noxious heat. Third, the heat threshold for all neurons ranged between 42 and 47 °C, which is consistent with the documented threshold for TRPV1 in an expression system.9 Thus, in isolated somata, heat-evoked inward currents in neurons from naive, nerve injured and inflamed mice were entirely due to activation of TRPV1.6 In contrast to the soma, heat responses at the peripheral terminal are less dependent on TRPV1. Recordings from skin-nerve preparations, where heat is applied at the peripheral terminal and responsiveness is recorded in the peripheral axon, indicate that heat-sensitive C fibers with thresholds ~ 42 °C remain partly intact in TRPV1 deficient mice8, 30, 63, 66 (and Stucky C.L., unpublished data), albeit to different degrees in different laboratories. The residual heat responses in peripheral axons may be mediated by heat-sensitive ion channels expressed only at the terminal region of the neuron or by non-neuronal cells in the skin. For example, keratinocytes express functional heat-sensitive channels including TRPV3 and TRPV419, 47 and recent evidence suggest that keratinocytes confer heat responsiveness to nearby sensory terminals via release of ATP.31, 40 Together, these findings indicate that future studies searching for heat transduction molecules should include a component where the sensory terminal remains in its native milieu such that interactions between non-neuronal cells in the target tissue and the sensory neuron membrane can be appropriately modeled and investigated.
When using isolated DRG somata as a model for the sensory terminal, the dissociation and culture procedure may intrinsically injure the neuron, thereby altering the composition and density of ion channels normally expressed by the soma membrane in vivo.29, 65 Nonetheless, using DRG somata as a model provides an important measure of the overall function of TRPV1 channels in the plasma membrane. Indeed, TRPV1 is expressed by many membrane regions of the sensory neuron, including central terminals in the spinal cord,44 peripheral axons of passage,4 the cell body and the peripheral terminals. In our study, DRG somata from all groups were processed in an identical manner, and therefore, the differences observed were likely due to the injury in vivo.
The somata of directly injured neurons no longer have axons that reach peripheral targets. Injured neurons frequently form neuromas at the cut end of an injured nerve. Increased expression of ion channels at the neuroma in the setting of an inflammatory milieu may contribute to spontaneous pain via spontaneous input to the spinal cord and/or sensitization of spinal cord neurons. It has been hypothesized that increased TRPV1 function may contribute to spontaneous activity via a reduction in the thermal threshold, such that in the setting of nerve or tissue injury, TRPV1 becomes activated at body temperature.52, 56
Our data add to a growing body of evidence that IB4 positive C fiber neurons play important roles in injury. The majority of cutaneous C fiber neurons that innervate the epidermis bind IB4.35 They are known to be nociceptors and respond to mechanical stimuli in situ.1, 17, 60 A variety of animal models of persistent pain indicate that IB4 positive neurons are highly sensitive and malleable to injury and upregulate their functional response properties in response to injury. For example, IB4 positive neurons increase TRPV1 function after peripheral inflammation.6 Interestingly, evidence now indicates that IB4 positive neurons provide input to a special pathway to the brain that is involved in modulating the emotional processing of pain.5, 69 Together, these data suggest that IB4 positive nociceptors may be involved in providing enhanced input to the emotional aspects of neuropathic pain.
Nerve injury pain is due to a mixture of effects from axon injury and inflammation. Inflammation is well-documented to increase TRPV1 function.8, 12 In our study, neurons from animals with sham surgery were very likely exposed to some degree of inflammation. This is suggested by a trend for increased TRPV1-mediated heat responsiveness in medium-large neurons from sham animals compared to naive controls (Fig 5A). In animals with SNL, the L3 adjacent neurons were exposed to minor injury and inflammation, whereas L4 neurons were axotomized and exposed to inflammation. We speculate that these differences in inflammation and axon injury between groups underlies our finding that TRPV1 function was significantly decreased in injured L4 medium-large neurons compared to sham surgery controls, and that the net effect of direct injury on myelinated neurons is decreased TRPV1 function. Thus, the overall change in TRPV1 function in myelinated neurons is due to the combination of inflammation and nerve injury. In different animal models of nerve injury, if inflammation is predominant, TRPV1 function increases; if injury is predominant, TRPV1 function decreases.
In behavioral experiments, L4 SNL induced heat hypersensitivity in the hind paw that was completely reversed by acute intraperitoneal injection with the TRPV1 inhibitor A-425619. In the days following L4 SNL, the plantar paw presumably loses innervation by the L4 afferents but retains innervation by L3 afferent terminals. The heat hypersensitivity is not likely due to sensitization of L3 adjacent neurons because we found no change in TRPV1 function in neurons from the L3 ganglia. Instead, we observed increased TRPV1-mediated heat responses in IB4 positive neurons from the injured L4 ganglia. We propose that increased TRPV1 activity in injured IB4 positive neurons, possibly due to reduced TRPV1 heat threshold and activation by body temperature, contributes to enhanced spontaneous activity in these neurons that subsequently sensitizes spinal cord neurons to respond more to input from intact L3 afferents activated by peripheral heat stimuli.
Knowledge about nerve injury-induced changes in TRPV1 function has important clinical implications. First, injury-induced upregulation of TRPV1 function provides rationale for treatment strategies that inhibit or desensitize TRPV1. Second, identifying specific neuronal subtypes that exhibit increased TRPV1 function may lead to treatments that specifically target these neuronal subtypes. For example, selective inhibition or elimination of IB4 positive C fiber-type neurons during neuropathic pain may help alleviate negative emotional aspects of chronic pain, but leave intact the acute, protective nociceptive responses necessary for safe interaction with the environment.
This work was funded by NIH grant NS40538 to CLS. We thank Dr. Aniko Szabo, Ph.D. in the Biostatistical Consulting Center at the Medical College of Wisconsin for assistance with statistical analyses.
Perspective: TRPV1 function is upregulated in IB4 positive sensory neurons and TRPV1 is responsible for the behavioral heat hypersensitivity in the spinal nerve ligation model. Since IB4 positive neurons may contribute to the emotional perception of pain, TRPV1 antagonists, targeting both sensory and affective pain components, could have broad analgesic effects.
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David Polkinghorne Western District pushed reigning premiers Central to a 4-all draw, but coach Adam Baxter says Wests still need to prove to themselves and the competition they are worthy of being...
Canberrans hoping to get picked up in AFL women's draft
David Polkinghorne Belconnen defender Heather Anderson is unsure what she's more excited about - the chance to get picked in the women's AFL draft on Monday or playing on the MCG in front of 90,000 people on Anzac Day.
Nick Salter kicks 14 in Ainslie's big NEAFL win over Eastlake
David Polkinghorne Ainslie forward Nick Salter is making a habit of kicking big bags of goals against Eastlake, his haul of 14 almost double the Demons' total score at Alan Ray Oval on Saturday.
Orica-GreenEDGE's Mathew Hayman backs Michael Matthews to win Amstel Gold Race
David Polkinghorne Orica-GreenEDGE captain Mathew Hayman says Michael Matthews' disappointment and hunger will help drive him to win the Amstel Gold Race in Holland on Sunday.
Canberra wrestler Nick Verreynne after Australian title
David Polkinghorne Hopes to put Olympic pain behind him.
Eastlake recruit Nick Fothergill wins NEAFL rising star award
David Polkinghorne Ainslie plays Eastlake in first Battle of the Bridge.
Criterion greatest Black Opal winner, but Takedown could deliver Champagne celebration
David Polkinghorne Black Opal future looking rosy and producing champion horses.
Canberra golfer Nikki Campbell's pregnant pause for Australia Classic
David Polkinghorne Golf shoes and pregnant, that's how Canberra's Nikki Campbell will tackle the inaugural Australia Classic in Sydney on Friday.
Carl Valeri dedicates wonder goal for Melbourne Victory to dad
David Polkinghorne Dedicates stunning goal to father.
Coach John Fowlie says missing worlds a 'blessing' for Canberra swimmer Alicia Coutts
David Polkinghorne Swimming coach John Fowlie says five-time Olympic Games medallist Alicia Coutts missing this year's world championships squad could be a "blessing" ahead of next year's Olympics in Rio de Janeiro.
Kimberley Wells wins back-to-back stages in Adelaide Tour
David Polkinghorne Canberra cyclist Kimberley Wells has finished the Adelaide Tour with back-to-back sprint wins in the National Road Series on Sunday.
Canberra Olympic recruits announce their debut with matching hat-tricks
David Polkinghorne Canberra Olympic coach Frank Cachia said recruit Philippe Bernabo-Madrid came to the club with a point to prove and the former Canberra FC striker did exactly that in emphatic fashion with a...
Canberra cyclist Mathew Hayman to lead Orica-GreenEDGE at Paris-Roubaix
David Polkinghorne Bone-jarring sections of cobblestones are just a fall away from broken bones and rain can turn the race known as the "Hell of the North" into a muddy nightmare, but that's what Canberra cyclist...
Belconnen United win 4-2 after Gungahlin United player sent off for headbutt
David Polkinghorne Gungahlin United appeared on course to a dream start in Canberra's National Premier League until a Liam Walsh headbutt and resulting red card proved to be a "game changer".
Australian football
Ainslie and Eastlake get off to losing start in NEAFL
David Polkinghorne Ainslie coach Chris Rourke is expecting a tough Canberra derby next after both ACT teams were outplayed in their NEAFL season openers on Saturday.
Belconnen United form alliance with A-League club Sydney FC
David Polkinghorne Belconnen United will kick off its premiership defence by creating a genuine pathway to the A-League.
Canberra clubs Ainslie and Eastlake expecting tough new NEAFL structure
David Polkinghorne Ainslie coach Chris Rourke says the Tricolours "expect to play finals" in 2015.
Just A Blur to back up in $1 million Queen of the Turf Stakes
David Polkinghorne Just a Blur given a crack at country's best.


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PRLog - Jan. 16, 2014 - CHENNAI, India -- As the title suggests, it involves collecting data from the field which is primary in nature. Collecting the data in this mode is very challenging as it requires interaction with a lot of stake holders and their perception in context of research matters a lot. The challenges are difficulty in:
Field data collection
Engaging the participants
Keeping the motivation high
So, these are the baby steps in reflecting to know what is happening or what exactly they are expecting. But the field survey will enable the researcher to identify the area of concern including the various feasible options to solve. While collecting data from the field, you can't go to anybody and ask questions relevant to your research. You must analyze the respondents from a targeted population to get the reliable answers. Therefore, you must exhibit ethical traits and considerations such as:
Protecting the dignity and welfare of the respondents
Respect for those who decline to participate
Invasion of privacy
Hence, the pros and cons of respondents must be taken in account in sampling. When sampling, you must analyze the field of survey (http://www.phdassistance.com/
Collecting data from the field is no doubt a herculean task but time consuming. One can't rule out the issues and obstacles but it requires strategic planning to deal with. First thing is the researcher must have sound grasp over the subject with statistical bend of mind. Expertise along with the statistical tools must be used effectively to collect data. Focus is collecting valid data to make a solid inference. Therefore, framing the questionnaire is obviously a tool; you must be well versed to design it. Second thing is the way to approach the respondents. You must exhibit the ethical manner so as to expect better reply. These are the generic issues discussed but you must visualize what could be the other potential issues. If you have little idea for data collection; then you could refer the task to somebody who has more experience and expertise. In this case, we would like to provide assistance to deal with the issues of data collection in any area of research. We have the experts having experience, expertise, and credibility, who could add more insights for your data collection requirements. The experts are committed to provide ways and means in order to obtain accurate and valid data.


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Discover a selection of top forums balkan explorer on the internet. Come to share your passion on forums balkan explorer and meet other fans of balkan explorer. Then, why don't you create your own forum of balkan explorer ?
Ljubitelji NFL-a iz Srbije.
fans, serbia, ljubitelji, nfla, srbija, #balkan, americki, fudbal, ncaa, football, koledz, saaf, crna, gora, slovenija, hrvatska, makedonija, forum, tacdaun, vukovi
You are a POKEMON striving for adventure and dream of becoming a famous explorer. But you can't do it alone! You decide to get your start in the Zebstrika Army Guild
zebstrika, army, guild, jacobasalways, strikingly, brave, pokemon, striving, adventure, dream, famous, #explorer, can't, alone!, decide, start, mystery, dungeon
Balkan Public 22.214.171.124 Join
iphone, free, virtual, forum, enjoy, amazing, from, comfort, your, internet, #explorer, browser, thepublicsdomain, com/virtuali
Free forum : First Mitchel Musso Balkan Forum
Free forum : BALKAN EVONY TEAM


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Originally Posted by akfandisney I Know what Al said and my information is come from Disney store manger castmember. I know that that all castmember information is not true.
Also the castmember say that old candy make place by the barn will become house for the horses.
My think is that the new buliding may be both candy make and bakery. Not sure about the new building but I'm pretty sure the production kitchen for candy making is in DCA at the old Hollywood and Dine kitchen.
Originally Posted by mycroft16 mrfantasmic beat me to it.
Here is the link to when Al announced the news back on Jan 8, 2008. http://miceage.micechat.com/allutz/al010808d.htm (Section called: In the Oven) Wonderful isn't it!? Disney is going to save so much money, and there's nothing like having fresh bread smell around the parking lot! =D
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Pecan Crusted Chicken
Fresh Spinach and Fettuccine Alfredo
Apricot-Stuffed Chicken Breasts
Garden Medley Alfredo Chicken Skillet
Wild rice and mushrooms bring a rustic flavor to an updated version of the classic chicken and pasta dish.
If you can't find chicken broth with garlic, make your own by finely chopping 2 cloves of garlic and stirring it into regular chicken broth.
Two cups refrigerated Alfredo sauce can be used instead of the jarred sauce.
For the best flavor and texture, make sure the wild rice has cooked long enough to "pop" open to show its white insides. You may need to add more water during cooking.
© 2015 ®/TM General Mills All Rights Reserved


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We can't seem to find the page you are looking for. It's possible the content has expired or has been moved. Perhaps if you use the sitemap below or the search box above, you might have better luck.
UPDATE: Lloyd scores 3 and US defeats Japan 5-2 for World Cup title


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ALA Midwinter Spotlight: All Our Coverage
Our overview of ALA's 2015 Midwinter Meeting, January 30 to February 3 in Chicago.
The Top 10 Library Stories of 2014
PW takes a look back at some of the library stories that captivated the publishing world in 2014, and what they portend for 2015
Books and Browsers: Building the Nimble Library
Building the nimble library
Can Open Access Save the Scholarly Monograph? ALA Midwinter Spotlight
Scholarly monographs, long the gold standard for scholars in the humanities and social sciences, have been in a downward spiral for some time.
We Build The City
In a popular post last month on Motherlode, the New York Times's parenting blog, Luisa Colón made a surprising admission: she doesn't like to take her kids to the library anymore.
Digital Audiobook Lending Is Finding Its Groove
By all indications, digital audiobook lending is growing in popularity among library patrons across a number of demographics.
Check It Out with Peter Brantley: New Standards for E-book Distribution
In a fast-changing industry like publishing, the simple things are often the most useful.
Tips for Librarians at The Guadalajara Fair: Spanish-Language Publishing 2014
The Guadalajara International Book Fair (FIL), the most important publishing gathering in Ibero-America, will be held this year from Saturday, November 29, to Sunday, December 7, with thousands of attendees from around the world.
The Fault in Our Stars
Every few months the notion of celebrity librarians (celebrarians?), sometimes embarrassingly referred to as "rock star librarians," appears on our radar.
EveryLibrary Gears Up for November Elections
EveryLibrary is gearing up for a big election day on November 4.
Aspen Institute Report Urges Public Library Transformation
A new report from the Aspen Institute looks at the importance of U.S. public libraries, and how they can change and improve to meet new roles in the 21st Century.
3M Library, B&N Offer Nook Lending Program
3M Library Systems is partnering with Barnes & Noble to allow library systems to purchase the Nook GlowLight e-readers directly from the retailer to lend to their patrons.
Michael Kelley: How Libraries Preserve E-books
Librarians caution that we are tumbling toward a digital memory hole in which large portions of our literary heritage could one day be lost altogether.
JukePop Hits Kickstarter for Funding
JukePop, an e-book platform that provides book discovery for self-published authors by offering libraries and traditional publishers the ability to identify popular indie works, has launched a Kickstarter campaign to raise $15,000.
How Strategic Library Initiatives Can Fail Miserably
Hard-learned lessons from the New York Public Library system that can help large and small libraries across the country.
New Pew Report Explores Young Americans' Library Habits
A new report from the Pew Research Center Internet Project offers mixed news for public libraries.
Check it Out with Michael Kelley: ALA Moves on Legislative Goals
Much of the media coverage of libraries in 2014 to date has centered on the modest progress made on the e-book issue. But 2014 has been a year of significant progress for libraries in the public policy sphere, with a number of library initiatives advancing.
Floating Library Opening on the Hudson
Beginning September 6, New Yorkers will get a chance to visit a floating library, housed in an historic steamship berthed at Pier 25 on the Hudson River.
How E-book Subscription Programs Will Affect Libraries
With the launch of Kindle Unlimited (KU), Amazon's subscription service, consumer-oriented e-book subscription services are beginning to look like a coming thing.
The P&L Sheet: Against "Curation"
I knew the word "curate" had successfully shed its musty history—cluttered with Etruscan vases and dioramas of American bison—when I got an email from Brooks Brothers a few years back announcing that it had "carefully curated" a collection of shirts for my consideration.


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Fox Bend Golf Course, Oswego's own welcoming and challenging public course, boasts plush fairways, mature trees, and challenging greens. This 6,890 yard, par 72 course tests skills of all levels with 34 well-positioned bunkers and water coming into play on 11 holes.
Voted a Five Star Course by Chicagoland Golf magazine, Fox Bend has hosted three Illinois Opens, a 2010 and 2011 USGA Qualifier, 2012 Illinois Amateur Qualifier and will host 2015 State Am Qualifier in June.
Book your golf outing or our pavilion for a special event. You won't find a better value!
Foot Golf at Fox Bend
Begins May 4th
View Footgolf Here.


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- Berries
- 1 pint blueberries washed and dried
- 1 pint blackberries washed and dried
- 1 pint raspberries washed and dried
- 3 tablespoons sugar substitute
- 1 vanilla bean halved and seeds removed
- Whipped Cream
- 8 oz whipping cream
- 1/2 teaspoon vanilla
- 2 tablespoons sugar substitute
Servings: people
Carbs Per Serving:25g
Prep Time:5minutes
Cook Time:20minutes
- Heat your oven to 450 F.
- Place the berries on a sheet pan and sprinkle with the sugar substitute and vanilla bean seeds. Toss to combine. Roast in the oven for 20 minutes.
- Cool slightly before serving.
- Put the cream in the bowl of an electric mixer with the whisk attachment.
- When the cream starts to thicken, add the sugar substitute and the vanilla.
- Continue whipping until stiff peaks form. Serve with the berries.
For the nutrition calculation, we used Splenda Granular. If you use real sugar instead of substitute, the recipe has 30g of carbs per serving.


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I tried yoga 14 years ago for the first time and found that it was the most uncomfortable, unpleasant, difficult exercise on the face of the planet Earth. After one hour of Sun Salutations and other moves my body was aching badly and I was in my 20s at the time.
Along the years, I've tried yoga here and there and have never really been able to get into it.
A few years ago, I discovered Pilates and took quite a liking to it, but found out that it had a lot to do with the teacher. Paula is a 20-something blonde who has the ability of turning any body into a flexible-power-house and I liked that a lot.
That said, given the odd times of Paula's Pilates classes, I never was able to stick to them because I couldn't always make it.
The objective for me in practicing yoga or pilates was never to be connected with my inner self or to experience the life of a Buddhist Monk, but it had everything do to with the fact that I've never found that I was the most flexible or limber person and I felt that I needed to add that aspect of a workout to complete my exercise routines.
I'll also admit that seeing Madonna, when she was in her late 40s, able to do things with her body that was previously reserved to Cirque du Soleil performers was very impressive and I wanted to be able to increase my flexibility.
A side note on Madonna, have you seen her in those Louis Vuitton adverts? Some feel she doesn't look sexy because her body is too toned, but I think that Madonna is a trailblazer when it comes to redefining what a 50 year old's body should look like (who says that being in your 50s automatically mean that you're out of shape). I know she's a devotee of yoga and it shows. I will shamelessly admit that I aspire to have Madonna's body in my 50s! The woman looks incredibly hot and super sexy.
Check out the Louis Vuitton ads (featuring sexy Madonna in her 50s) if you've not seen them yet:
Here's the video showing the making of the Louis Vuitton ad with Madonna:
One month ago, I had an epiphany at 37.77 degrees Celsius (100 degrees Fahrenheit) and discovered the joys of hot yoga (aka Bikram yoga)!
I had heard of hot yoga, but the first thing that came to mind was smelly people crammed in a small room sweating like crazy. The hot yoga room is indeed fairly small, it's always a full class, there is very little room between participants and we all sweat, but there is no bad smell!
Hot yoga is very similar to your traditional Hatha yoga, but the room it very warm (and there's a good reason for that and I talk about that in the next paragraph).
There are many benefits to hot yoga that you won't find in traditional yoga:
* Hot yoga increases your body's ability to burn fat and build core muscles.
* Your body can more efficiently oxygenates blood, tissues, glands and your other organs.
* Hot yoga's optimal humidity improves the immune system and rejuvenates the skin. * Sweating by performing exercises in a hot room promotes detoxification. * Sweating also heals and cleanses the body from the inside out. * Hot yoga releases tight/tense muscles and relieves back pain
If you've tried yoga in the past, but you it didn't stick with you, I'd highly encourage you to try hot yoga because you will inevitably feel the benefits in your body, joints, posture and I guarantee that you'll have an incredible night's sleep!
Hot yoga is as popular as spinning classes and I'm sure that you'll find classes at your gym or you'll find a plethora of yoga studios in your neighbourhood that offers the classes.
First photo by evanbdudley
Photo of Madonna are from the new Louis Vuitton ads


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Let's see the summary of AutoCAD 2016 new features. There are some interesting enhancements in this release! Do you like it?
Featured Posts
Latest articles
How to create custom AutoCAD dimension arrow
Make your AutoCAD drawing looks fancy by using custom dimension arrow. Follow our guide here!
Do you remember your first time using AutoCAD?
First AutoCAD was released in 1982. We heard interesting stories about the old days, first time using AutoCAD. Do you have story to share? It can be first time using it in 80's or just yesterday. Tell your story here!
6 External Tools that came with AutoCAD installation
Do you notice that there are 6 external tools come with AutoCAD installation? They are relate to AutoCAD, certainly. But what do they do?


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Movie fans brave drizzle to enjoy animated adventure this lunchtime — full weekend movie listings here
Cut list down to 5 family and five childrens' movies
They are Kirkwood Hospice and Huddersfield Town Foundation
The Grove tops chart on both TripAdvisor and Examiner survey
Protestors from across Kirklees call for 'brownfield first'
Huddersfield is due to get its own rooftop bar — similar to these high-in-the-sky havens
Police were seen escorting Leeds fans from St George's Square ahead of today's Town game
Here's our review of the second half of 2014 — in 16 exciting videos
Here's our review of the first half of 2014 — in 14 exciting videos
The station is actually more than 150 years old - but we start our pictures in the 1890s
Timelapse video was shot from the Britannia building - whole day in less than TWO minutes!
Plenty of Christmas cheer from lights switch on in Meltham to mulled wine in Beaumont Park
Have you put your tree up yet? Send us a snap
Revellers packed St George's Square for top hip-hop DJ set to thrill sci-fi lovers
DJ Yoda took thousands of revellers on a nostalgic trip through sci-fi time
A quick-paced Riverdance played brilliantly by Hade Edge Band was the finale to the family section of the festival
The rain held off as the festivities kicked off with the Spark! drummers, who paraded into the square to their own energetic rhythms
The award-winning DJ will headline the festival with a sci-fi themed set
What's on, event previews, where to park and what roads are closed - here's all you need to know about Saturday's festival
Winter warmers, food from around the world and more in St George's Square
Huddersfield Examiner
For the latest local news straight to your Twitter, Facebook feed or e-mail inbox.
Is there something happening in your area you would like us to report on?
The Huddersfield Daily Examiner is the leading morning title read throughout the Huddersfield and Holmfirth areas.
With a daily circulation of 18,241 (ABC July-Dec 2012) and every issue read by 49,374 (JICREG 1/10/12) we are the area's favourite and most trusted newspaper.
Our www.examiner.co.uk website is the number one local news website in the area with 250,000 unique users and almost 2,000,000 page views a month*, 44% of who are in the desirable ABC1 socio-economic groups.
For more advertising contacts email email@example.com or call us on 01484 431111.
*Source: Omniture (UK 6 month average Jan-Jun 12)
Editor, Huddersfield Daily Examiner
Roy Wright was appointed editor of The Huddersfield Daily Examiner in 2002. Prior to this he was assistant editor of The Liverpool Echo for three years. He has also worked at the Hull Daily Mail as well as a number of Press agencies.
Roy grew up in Burnley, Lancashire and has worked in the local newspaper industry for 25 years.


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The Restraint Control Module (RCM) is the "brains" behind the airbag system. Portions of the module are coated to help prevent moisture intrusion. The coating can crack and when exposed to humidity the printed circuit board may short circuit. This could cause one or more of the airbag system components not to function as expected in the event of a crash, increasing the risk of personal injury. Replacement parts are currently not available, a second notification letter will be sent when they are and dealers will perform the necessary repairs.
Ford Escape Recalls
The coolant pump for the hybrid system may fail, resulting in the hybrid system shutting down, and the vehicle losing power. Dealers will inspect and replace the original motor electronics coolant pump free of charge to correct the concern.
The engine may hesitate while driving and can stall due to a wiring issue to the manifold absolute pressure (MAP) sensor in the engine management system. Dealers will replace the wiring splices in question free of charge to correct the problem.
Due to a software issue that affects operation of the torque sensor, the electric power steering may stop functioning. This would make it difficult to steer the vehicle. Dealers will reprogram the power steering control module and instrument cluster module free of charge to resolve the concern. Dealers will also replace affected electric steering components as necessary upon inspection free of charge.
Programming errors in the software for the restraint control module (RCM) can result in delayed deployment of the side-curtain airbag. Delayed deployment in certain rollover circumstances would increase the rick of personal injury. Dealers will reprogram the RCM to correct this concern.
The recliner mechanism for one or both front seats may have been improperly welded to the seat frame. As a result, the seatback may become loose or lean, increasing the risk of injury in the event of certain types of crashes. Dealers will replace the affected seatbacks to correct this concern.
The front suspension lower control arm may corrode at its forward attachment, and may separate from the vehicle. Dealers will install a newly designed reinforcement cross-brace free of charge to resolve the concern.
1.6L engines can develop a localized overheating condition that can result in a cracked cylinder head and engine oil leak. Leaking engine oil may contact a hot surface increasing the risk of an under hood fire. Dealers will modify the engine shielding, cooling and control systems to correct this concern. For some vehicles, this recall is in addition to a previous recall, they will need to be reinspected and have the new remedy completed.
Vehicles with a 1.6L engine previously recalled for a fuel line concern (NHTSA #12V-336) are being recalled again because the fuel line replaced previously may have been installed incorrectly. A fuel leak can develop, increasing the risk of a fire. Dealers will inspect the suspect fuel line and replace it as necessary to correct this concern.
The left rear door lock assembly was not manufactured correctly and as a result, the child lock may not engage properly. When the child lock is not engaged, the door can be opened from the inside, increasing the risk of injury to an unrestrained child. Dealers will inspect the the left rear door latch and replace it as necessary.


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Rated 3.9 out of 5 by 233 reviewers.
Rated 4 out of 5 by BLITZ COMMENTS NOTICED THE HOTEL IS GETTING IN THE NEED OF A FACE LIFT IN AREAS...RUGS ECT...POOL IS TO COLD...ALSO BEDS A LITTLE TO HIGH... June 28, 2015
Rated 5 out of 5 by Faris123 Good hotel I had a good stay at the hotel . David at the front desk was so helpful and would like to thank him. The room was clean, comfortable and well equipped. June 28, 2015
Rated 5 out of 5 by PilotPete1980 Excellent one night stay. The Bristol Place offered me just what I was looking for. A quiet, relaxing night before a big wedding weekend in Ottawa over May long. I checked in late and was greeted promptly. The suite was large and the little extras (the snack tray/water) were more than appreciated. The bed was amazing. Awesome shower head. I had the pool to myself in the A.M. and loved it. Warm water and lots of towels. For the CAA price I paid which included WIFI I would stay here again. June 10, 2015
Rated 5 out of 5 by Flyingtailor My May 2015 Review Having stayed here at least 10 to 15 times since my last review what more can I say. My favorite two services are great front desk and the airport Shuttle amazing fast pick up and delivery. May 20, 2015
Rated 3 out of 5 by supatight80 good airport hotel choice This property needs some major renovation... Things were somewhat falling apart at the westin workout room they allocated me into... May 14, 2015
Rated 5 out of 5 by Paul007 Friendly and helpful Front desk Staff I made a last minute booking as I had to stay overnight for next day meeting. Quick check in, Sharon upgraded me to a deluxe king room, for being a Starwood Prefered guest. Nice,spacious and clean room. Had a good night sleep. Breakfast was refreshing at the restaurant.. March 30, 2015
Rated 2 out of 5 by KJB3 Needs Updating The rooms were clean but the hotel itself is dated and is showing it's age. The hallways at the room levels were especially drab and dated with cracked ceilings on most floors. March 27, 2015
Rated 1 out of 5 by GSONT Give this one a pass Tired property. Problems with noise and smoke smells in non smoking rooms. Efficient shuttle service from airport March 23, 2015


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The GPT specification clearly states that if the MBR contains no protective GPT entry (one with a type code of 0xEE), that it's not a GPT disk. Your disk has no type-0xEE MBR partition, so it's technically an MBR disk, not a GPT disk. Furthermore, since you can boot Windows, Windows is clearly using the disk as an MBR disk, so that's what it is from a functional point of view. At best, the GPT data duplicate your existing MBR partitions. At worst (and this is more likely), they provide contradictory partition information that, if some tool were to try to use it, could end up damaging your real partitions.
Your immediate problem is that libparted is getting confused by the valid MBR and leftover GPT data. You've got to clean out the leftover data.
It's currently in MBR format. The problem is that whatever utility you used (probably the Windows installer) did an incomplete job in converting it back, in that it didn't wipe all the GPT data.The whole GUID makes sense now. About a month ago I tried to do a hackintosh and couldn't get it. I decided to just reinstall Windows, so did I forget to format my hard drive back to an MBR?
It's unclear what was unable to access your Windows 7 files. Was this the Ubuntu installer in "live CD" mode? Something else? In a normal Linux installation, it is possible to access Windows files. I suspect you either didn't know how to do this or there was a problem related to the leftover GPT data. In any event, I recommend you put this question off until after you've installed Linux, with one caveat....Also, I noticed that I wasn't able to access my Windows 7 files. When Ubuntu installs, will I be able to?
It's unwise to regularly access any OS boot partition from another OS. In this specific case, Linux doesn't understand the security features that Windows uses on its filesystem, so it's easy to accidentally trash your Windows setup as an ordinary user in Linux. Also, although Linux's NTFS driver seems pretty reliable based on anecdotal reports, I find it hard to believe that it's as reliable as Windows' own NTFS support.
Thus, I recommend that when you repartition, you create a separate FAT or NTFS partition for shared data -- any files you might want to access in both OSs (MP3s, photos, etc.) or files you might want to transfer between OSs (if you download a Windows file in Linux, say). Mount and use this partition in both OSs, but when you're in Linux, do not mount the Windows C: partition. That'll keep the OS installation itself safe from damage.
If you wipe the GPT data, they will trouble you no more, unless you mess around with tools to convert the disk back to GPT form. I'm not sure what you mean by "installing any other files."Finally, if I get Ubuntu installed, will I ever have any more problems with the GUID partition table or installing any other files?


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They feel bad, so they found you these other deals to make up for it.
Select Local Merchants
Sonoma Medi SpaRead MoreThe team at Sonoma Medi Spa help clients with anti-aging and rejuvenating therapies including dermal fillers and clear skin with chemical peels. The spa's number of laser treatments are performed by Medical Aesthetician Lori Hetland and overseen by Dr. Peter Spanganberg. The skilled medical team can also smooth sagging eyelids and foreheads with tasteful surgery.7400 S Power RoadGilbert, AZ US


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My old username was carbednotch
My SRT has such sloppy steering bell cranks... The aluminum steering pivot bushings need replacing also.
The aftermarket has a nitro rustler alumin steering turnbuckles with ball bearings but I found nothing specifically for the srt. Will the nitro rustler steering fit my srt? I really want ball bearings instead of the plastic bushings


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Chevrolet Silverado 1500 Problem Report
Chevrolet Silverado 1500 Intermittant Failure to Start
(170 reports)
Exactly same problem as described by Schmackster1952. Problem is totally intermittent, but frequency of occurrence seems to be getting higher. Battery, charging, and all other electrical system components seem OK. I have tried moving shift from park to neutral; took key in and out of switch; nothing seems to be related. Is there any switch, relay or sensor that could cause the truck to "think" it is not in park, or the engine is already running, or whatever else might keep it from engaging the starter motor. When it decides to act right, all is will. Anyone else found a solution? - me2942
90 people like this
Will turn key and it is like no power from battery to starter, but have lights, radio, etc. Turn key again or sometimes multiple times, to no avail. Then, turn it one more time and it starts. Then the next 15 starts, no problem. Sometimes hot engine, sometimes cold. UPDATE: 2-6-14; I sold the truck to my father. He changed the starter. No problems since March 2013. - Schmackster1952
58 people like this
Chevy silverado 1500 2004. The same problem, random intermittent starting. I had had this problem before about 5 times and each time I disconnected and reconnected the negative battery cable it started fine. This time that didn't work. So I swapped the starter relay in the fusebox under the hood with the other identical relay, no luck. I crawled underneath the truck and tapped the starter with a hammer, no luck. I jumped the truck, no luck. I did hear the solenoid clicking. The door was open and the dome light was on. When I turned the key to start the dome light did not flicker or dim in any way. Also, when the key was on, the battery voltage read about 12. When I turned the key to start, the gauge dropped down to 0 and stayed there until I released the key back to ON. Also, I keep the headlights on automatic with the selector switch. They stayed on while the key was in the on position with the truck in park, and died when I turned the key to start. The check engine light was on when the key was On and I think stayed on when I turned the key to start. That was yesterday. Today was totally the opposite. I flicked the key to start this morning and heard it try to start. I flicked it again and it started. I did not jump or charge it. I drove it for about 15 minutes. The battery gauge showed 14 volts while driving, so the alternator was good. The headlights went off when I turned the key to the on position with the truck in park. Yesterday they had stayed on. The dome light did NOT come on when the door was open. I turned it on with the switch. The check engine light went off immediately after I turned the key to start. Yesterday it stayed on. When starting, the dome light flickered (I had turned it on manually today) , as it should. Yesterday it had not flickered at all when turning the key to start. But, yesterday the battery gauge went to 0 when trying to start. Today it went down just a bit while cranking, went to 0 for a half second, and jumped back up to 14 when it started. - Visitor
48 people like this
Same problem reported by others. I especially agree with comments by 'visitor' who described the situation in great/accurate detail. I believe it is related to 'anti-theft' or 'alarm-set' circuitry. If I repeatedly lock and unlock the doors, either at the door controls or with the remote, it will start after some manipulation. I find it hard to believe that GM has not found the source of the problem and the fix....since many owners are reporting the same issue. - Largobob
31 people like this
Turn key to start nothing happens keep trying and it will start and it might not do it again for a week - Visitor
21 people like this
just randomly wont start for the first time in the morning.....just walk away for part of the day or next day, and it starts. Different components are powered up each time....sometimes lights work great and horn but the next time something else works or doesn't work. It does seem to happen about once a month. Someone told me it could be on-star related. Any other ideas?....sad for a new car. Back to Ford I go - Visitor
19 people like this
One more thing about my 2004 silverado. I already wrote a book, but forgot to mention that the antitheft icon would stay on yesterday when the key was on and it wouldn't crank at all. However, today, when the truck will crank and start, the antitheft icon goes off after a few seconds with the key on and stays off while the truck is running. It never flashed either today or yesterday though. And I don't know if the antitheft system disables cranking or starting or both. - Visitor
19 people like this
Pretty much same as every one else, mine doesnt even crank. the starter relay doesnt click, fuel pump relay does cycle, all lights go out as if it is going to crank but doesnt. prior to this it would intermitantly start and immediatly stall but start right back up. - Visitor
13 people like this
same as others - Visitor
12 people like this
pretty much same as everybody else looking for answers but no one seems be be able to find it - Visitor
11 people like this
I have the same problem and I am glad I am not the only one. I own a 2007 Silverado with a 4.8 *newer body style. I have owned this truck for a year now and both winters I have had the same problem. Power works except when you turn the key nothing happens. Last year it happened a total of 5 times and it was very weird to me how it would act. At first when it would not start I would go to work and come home and it would start. Other times I would go in the fuse box check it to find no blown fuses but just a little corrosion on the starter relays but not enough to cause concern. I checked all the other connections and had nothing loose than I would go to turn the ignition and it would fire right up. Thankfully it would always eventually start. Fast forward to now and last night it happened again. So here is my theory. 1. Cold weather is probably most of the problem. 2. Don't wash your truck like me in the bitter cold because I now have a layer of ice on everything under the hood. I am thinking it could be causing a bad connection somewhere. 3. I have noticed the anti left icon stays on which never happens only when it does not want to start causing me to think this is shutting the power off to the ignition but must be a timer?? - Visitor
5 people like this
Truck wants to turn over but will act as if is starving for fuel,wait ten minutes, it start. Been happening frequently lately. I also was told it was the security system - Visitor
stopped truck filled up with gas went to restart no power to starter dash lights on until you turn key all the way to start all guages go to pre-start position had aaa bring me to dealer now i dont know what to expect - Visitor
Same issue as others described. Very intermittent. Happens once every 3 months or so, But lately has happened more frequently. - Visitor
same "no start" problem as reported by others on this blog. Original owner, 2002 Silverado LT 5.3 4 x 4 high mileage, very well maintained, Never had a starting issue until today. This particular -14 below and high winds cold snap is brutal . Brand new battery. Swapped 4 relays out, banged on the starter, Tried neutral start. Worked the shift lever up and down, blower and lights all work. Now I'm calling AAA......and going back to 1952 Buick. - tpgaynor
When I turn the key I get nothing. The only sound is when I turn the key back back to off it sounds like teeth rolling to line back up. Truck has been checked out twice but nothing found. It was sugested I try a different key as the other may be worn. The last time I waited 45mins before it started. Has anyone else had this problem? - Visitor
Happens more frequent - Visitor
Parked at home in the night after driving about 60 miles the same day. I decided to go out again, I turned the key and all of a sudden my truck starter didn't crank the engine or anything with the engine, I didn't feel anything vibrating from the engine except the visible guages on the dash made movements as always with the power on. Ok, I was turning the keys on and off 5 times only the guages that worked until after the 6th turned the keys over again then my engine started to crank and it started out fine. I was puzzled by it. After this very problem, it didn't happen to me twice or often. - Visitor
Same problem, found it to be due to corrosion on the top wire of the starter. Wiggled it around and truck will start. Cleaned nut and bolt and all is good. This worked for me but with the way there is movement of the wires in that area, it could also be a cracked or broken wire that makes contact sometimes, hence the intermittent starting. Area around starter is exposed to the elements and corrosion is inevitable, keep area clean and all should be good. - Visitor
I looked on another site and the first thing he said to check was ING E 10 amp fuse. I did switch this out but before doing so i noticed all the dust , and bugs even, that were in the fuse box under the hood. I blew it out before switching, which may have been the cure also. I recommend trying both. Running good for now, ill let you know if it flops again. - Visitor
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Fillet of Sole East Indian
|Sole fillets
|1 Pound
|Melted sweet butter
|2 Tablespoon
|Curry powder
|1 Teaspoon
|Ground cumin
|1⁄2 Teaspoon
|1⁄2 Teaspoon
|Freshly ground black pepper
|1⁄4 Teaspoon
|2 , rinded and juiced
|Dry white wine
|1⁄2 Cup (8 tbs)
|For sauce
|Sweet butter
|2 Tablespoon
|20 Medium, thinly sliced
|1 , finely chopped
|All purpose flour
|2 Tablespoon
|Dry mustard
|1⁄4 Teaspoon
|1⁄4 Cup (4 tbs)
|Light cream/Table cream
|1⁄4 Cup (4 tbs)
1. In a bowl, combine melted butter, curry powder, cumin, salt, pepper, lime rind and juice. Dip sole fillets into butter mixture.
2. Place sole in a large skillet. Pour wine over sole. Bring to a boil, reduce heat, and poach gently for 5 minutes, or until fish flakes easily with a fork.
3. Place sole in a shallow baking dish in a single layer. Reserve poaching liquid; set aside.
4. Preheat oven to 350°F (180°C).
5. Melt butter in a saucepan; add mushrooms and shallots; saute about 2 minutes.
6. Stir in flour and mustard. Whisk in milk, cream and 1/2 cup (125 ml) poaching liquid. Cook, stirring, over medium heat about 5 minutes, until thickened.
7. Pour over sole. Bake for 20 minutes, until sauce is bubbly.
Garnish with lime slices and chopped chives.


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March 13, 2014
Ask a Recruiter I Using Social Media in Your Job Search
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Glen Cove's Sea Faire
The inaugural Sea Faire at Glen Cove celebrates the city's revitalized waterfront and Independence Day as families enjoy a weekend of midway rides, treasure hunt with pirates, tours aboard the historic oyster sloop The Christeen and on Saturday night, Fireworks by Grucci. Some proceeds benefit the Glen Cove Youth Bureau. Videojournalist: Elise Apelian (July 6, 2013)
New LIRR cars short on space for crash technology Pedestrian dies after being struck by SUV on Route 111, police say Woman dies in Deer Park house fire Tall Ships arrive for festival in Greenport Summertime is here Working on the atomic bomb Suffolk cops demonstrate dangers of illegal fireworks Fire Island resort fire Extraordinary Seniors: Yaakov Kaminetsky Forecast: Partly sunny for July Fourth Thursday's puppy of the Day: Bruno Drag show at Cherry's On The Bay


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William Sheppee Saddler Sideboard
William Sheppee Saddler Sideboard
Weights & Dimensions
|Product Type: Sideboard
|Base Finish: English chestnut
|Top Finish: English chestnut
|Hardware Finish: Antique brass
|Distressed: No
|Gloss Finish: Yes
|Base Material: Wood
|Top Material: Wood
|Hardware Material: Brass
|Reclaimed Wood: No
|Solid Wood Construction: Yes
|Display Case: No
|Drop Leaf/Expandable: No
|Mirror Included: No
|Open Storage: Yes
|Drawers Included: Yes
|Cabinets Included: Yes
|Shelves Included: Yes
|Doors Included: Yes
|Leg Finish: English chestnut
|Leg Material: Wood
|Wine Rack: No
|Stemware Rack: No
|Commercial Use: Yes
|Eco-Friendly: Yes
|Product Care: wipe with clean cloth
|Country of Manufacture: India
|Assembly Required: No
About the ManufacturerFounded in London in 1988, William Sheppee is known throughout the world as the pioneer of Indian and Colonial Furniture, and a leading supplier of Indian antiques and architectural pieces. In 1996, William Sheppee relocated to a 36,000 square foot First World War aircraft hangar in Salisbury, Wiltshire and announced the establishment of William Sheppee (USA) Ltd. Based in the beautiful Tuscarawas Valley - the heart of Ohio's Amish country - William Sheppee's 25,000 Square foot (American) facility is centrally located, and therefore well positioned to service the American market.
William Sheppee Saddler Sideboard
Questions & Answers (1)
"Hi, is assembly required for this bench?" asked by Jasmin
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OUR SKU # WSP1049Saddler Sideboard is also sometimes listed under the following manufacturer numbers: SAD070


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Surviving Sandy
After the devastation of superstorm Sandy, we look back as Long Islanders reclaimed their lives. Newsday (Nov. 10. 2012)
Suffolk cops demonstrate dangers of illegal fireworks Fire Island resort fire Forecast: Partly sunny for July Fourth Puppy of the Day: Bruno Drag show at Cherry's On The Bay Forecast: Rain today, sunny for July Fourth Meet 'The Matz' sandwich Police investigate homicide in Central Islip Forecast: 80s temperatures for July Fourth weekend Willy Wonka-themed prom at Vandermeulen High School Police: Man stole cash from pizzeria tip jar Forecast: Clear skies for Eisenhower Park fireworks


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Member Since
Nov. 2009
Cooking Level
Cooking Interests
Baking, Frying, Slow Cooking, Mexican, Italian
Sewing, Hiking/Camping, Walking
Profile Link
Check out our collection of almost 200 grilled burger recipes.
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It's no secret that social media plays a major role in the way the hospitality industry connects with customers. There are, however, more ways for hotels to use social media than to just attract guests before they book. With creativity ...Read More »
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Canopy by Hilton to Launch 'Canopy by You' Campaign
MCLEAN, Va.—Canopy by Hilton has announced its first digital collaboration, Canopy by You. A three-month digitally-based program, Canopy by You reinforces the brand's commitment to locally-inspired design, food & drink, and art by engaging local tastemakers and curating neighborhood experiences ...Read More »
Mandarin Oriental Launches New Social Platform
HONG KONG—Mandarin Oriental Hotel Group announces the launch of Fans of MO, a new social platform where guests can share personal photos and notes about their favorite experiences at Mandarin Oriental hotels around the world. Visitors to the site are ...Read More »
Survey Finds Social Media Boosts Hotel Occupancy by 2x
PALO ALTO, Calif.—Hotel properties that actively engage with social media reviews grow occupancy at double the rate of properties that don't, according to a study released by Medallia. The study examines customer and business data from more than 4,400 hotel ...Read More »
Starwood's Digital Initiatives Connect with Guests
If you're looking to get your guests' attention, smartphones can be a good place to start. More than ever, guests are turning to mobile devices like these to research travel, book rooms, and interact with brands. From receiving instant directions ...Read More »


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Variable Speed Triblade Hand Blender 4 PC Kit
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Description: Variable Speed Triblade Hand Blender Kit:
- Four piece 380-watt hand blender kit with stainless steel blades and accessories
- Unique Triblade concept that delivers powerful, efficient and controllable blending
- Triple blade design
- Choose from variable speeds to ensure the most efficient chopping and blending of any ingredients
- Ergonomic grip
- Small castellated exit paths reduce risk of splashing
- Includes 25 ounce beaker, chopper and whisk attachments
- Measures 2 7/10 inches in diameter by 15 7/10 inches in height
- Wipe with a damp cloth; accessories are dishwasher safe
Manufacturer's one year warranty.
This product is manufactured in compliance with U.S. standards. If product is to be used outside of the U.S., use only with the appropriate outlet adapter and voltage converter for your country. Do not plug into an electrical outlet higher than 110-120V as this could result in fire and/or injury.
Material: Stainless steel, metal and plastic
Brand: DeLonghi
Origin: Imported


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Turbo Kids Jersey
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$30.98was $44.25
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Full List of Louis Theroux's Weird Weekends Episodes
This is an interactive, complete Louis Theroux's Weird Weekends episode list. You can even click on the episode numbers for more information in this list of Louis Theroux's Weird Weekends episodes. List is alphabetical, but can be sorted by any column. Episodes list includes links with episode synopses, when possible. Photos from the individual Louis Theroux's Weird Weekends eps are listed along with the Louis Theroux's Weird Weekends episode names when the information is available. Louis Theroux's Weird Weekends episodes from every season can be seen below along with information on the directors, stars and sometimes even fun facts about the Louis Theroux's Weird Weekends episodes, like shooting locations and original air dates. This list below has a variety of items in it, from Off-Off Broadway to South Africa. You can use the items in this fact-based list to create a new list, re-rank it to fit your opinion, then share it on Facebook, Twitter or any other social networks you belong to. (22 items)
- 1
Extras Dec 24 1998
- 2
Weird Christmas (Special) Dec 23 1998
- 3
Weird Christmas Dec 23 1998 Louis hooks up with four "weird" people, starring in the first season of his "Weird Weekends" documentary: fundamental Christian Randy James, reverend Robert Short, porn-star J.J.
- 4
Weird Weekends Extras Dec 24 1998
- 5
Televangelists Jan 15 1998 In Dallas, Louis meets TV evangelists Marcus and Joni Lamb, and joins a group of hardline Christians called The Family as they visit the Deep Ellum entertainment district.
- 6
Survivalists Feb 05 1998 Louis travels to the American North West, a place where survivalists, white separatists, and paranoid "freedom fighters" are arming themselves against the "New World Order". Meet
- 7
Porn Jan 29 1998 Louis strips off and plunges into the world of Porn. From the glossy high-end productions to the sleazy "gonzo" flicks, he gets intimate with the men and women who bang for bucks. But is it
- 8
UFOs Jan 22 1998 Louis has a close encounter with the community of hardcore UFO believers in the South West of America - but not even an illicit foray into the ultra-top-secret Area 51 military base and a garbled
- 9
Infomercials May 12 1999 Louis investigates the world of television informercials and tries to sell a paper shredder on television as an infomercial host.
- 10
Wrestling Jul 06 1999 How real is wrestling? How dangerous is it? Does Louis have what it takes to become one? Just some of the questions Louis answers in this adventure into the American subculture.
- 11
Swingers May 26 1999 Louis journeys to Southern California to investigate the weird world of wife-swapping. Is he man enough for the group room? And what do you do if you want to touch someone's primary erogenous zones?
- 12
Off-Off Broadway Jun 15 1999 Louis meets the hordes of out-of-work actors in New York as they try to land a part – and even goes on an audition himself, for a job on a Norwegian cruise ship.
- 13
Black Nationalism Jun 02 1999 American black nationalist groups have been branded anti-Semitic, homophobic, misogynist and even racist by the mainstream press. Louis Theroux goes to Harlem in New York to meet its proponents, and
- 14
Demolition Derby Jun 09 1999 Louis has to overcome his nerves to enter a demolition derby in car-mad Michigan.
- 15
Body Building Oct 16 2000 Louis travels to California, home of the body beautiful, to see if he can join the world of extreme body building. After working out with Guy Grundy, a leading amateur, he soon realises he is not cut
- 16
Hypnosis Sep 25 2000 Louis infiltrates the American self-empowerment movement to see if he too can use trance technique to unlock the secret to personal wealth and sexual attractiveness . But is it possible to be too good
- 17
India: Enlightenment Oct 02 2000 Louis travels to India to witness Westerners seeking enlightenment. In Goa he meets 57-year old American astrologer Deepak who studies meditation. He moves on to meet Mike, who is a follower of Swami
- 18
South Africa Oct 09 2000 Louis visits the whites-only communities that want nothing to do with a multi-racial South Africa and discovers a world of fire-breathing Boers ("Don't get cocky with me, my friend!") and
- 19
Thai Brides Oct 23 2000 Louis visits a Bangkok marriage agency where Western men meet Thai brides.
- 20
Gangsta Rap Oct 30 2000 Louis storms the Ghettos of the Dirty South with his own unique Gangsta Rap Flava. But how will the real-life players and pimps take to a rapper who enjoys fine wine and playing with kittens? And when
- 21
Law And Disorder In Johannesburg
- 22
Law And Disorder In Philadelphia


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HUF Arena Shoes, Black/ Gum
The Arena Shoes by HUF Footwear are a cup-sole, low-top skate shoe. The feature strategic stitching patterns in high-abrasion areas for a longer lasting skate shoe. They feature Premium Suede and Leather uppers.
ON SALE!!Sizes in Stock for Color: Black/ Gum
51.00% OFF!Reg Price: $72.00 Your Price: $34.95
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