Bodily Sensory Inputs and Anomalous Bodily Experiences in Complex Regional Pain Syndrome: Evaluation of the Potential Effects of Sound Feedback

Neuroscientific studies have shown that human's mental body representations are not fixed but are constantly updated through sensory feedback, including sound feedback. This suggests potential new therapeutic sensory approaches for patients experiencing body-perception disturbances (BPD). BPD can occur in association with chronic pain, for example in Complex Regional Pain Syndrome (CRPS). BPD often impacts on emotional, social, and motor functioning. Here we present the results from a proof-of-principle pilot study investigating the potential value of using sound feedback for altering BPD and its related emotional state and motor behavior in those with CRPS. We build on previous findings that real-time alteration of the sounds produced by walking can alter healthy people's perception of their own body size, while also resulting in more active gait patterns and a more positive emotional state. In the present study we quantified the emotional state, BPD, pain levels and gait of twelve people with CRPS Type 1, who were exposed to real-time alteration of their walking sounds. Results confirm previous reports of the complexity of the BPD linked to CRPS, as participants could be classified into four BPD subgroups according to how they mentally visualize their body. Further, results suggest that sound feedback may affect the perceived size of the CRPS affected limb and the pain experienced, but that the effects may differ according to the type of BPD. Sound feedback affected CRPS descriptors and other bodily feelings and emotions including feelings of emotional dominance, limb detachment, position awareness, attention and negative feelings toward the limb. Gait also varied with sound feedback, affecting the foot contact time with the ground in a way consistent with experienced changes in body weight. Although, findings from this small pilot study should be interpreted with caution, they suggest potential applications for regenerating BDP and its related bodily feelings in a clinical setting for patients with chronic pain and BPD

Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail

<div><p>While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.</p> </div

Inhibition of GSK-3 sensitizes melanoma cells to TRAIL-mediated apoptosis.

<p>A) A375 melanoma cells were treated with rhTRAIL at doses indicated on x-axis. Cells were concurrently treated with the GSK-3 inhibitor CHIR99021 (5µM) or DMSO vehicle control (v/v). Data represents the percentage of apoptotic cells measured by Annexin V-positive cells at 24 hours post-treatment by FACS. B) A375 cells were pre-treated with control siRNA or siRNA specific for β-catenin. 48 hours later, cells were treated with rhTRAIL (20ng/mL). 24 hours post-treatment, the percentage of apoptotic cells was determined by AnnexinV binding using FACS. Data represents mean percentage of apoptotic cells (+/- SEM). P-values were calculated by one-way ANOVA with a Tukey’s post-test analysis.</p

WNT3A sensitizes melanoma cell lines to TRAIL in a β-catenin-dependent manner.

<p>A) A375 melanoma cells were treated with indicated doses of rhTRAIL for 24 hours in the presence of WNT3A or control L-cell (L) CM (10%). Apoptotic cells were detected by Annexin V binding assay using FACS. Representative FACS histograms with Annexin V-positive gates are shown. Percent apoptotic cells are indicated. B) A375 cells were treated with indicated doses of rhTRAIL in the presence of WNT3A or L CM (10%). Data represents mean % apoptotic cells ± SEM as determined by Annexin V positivity at 24 hours. An (*) indicates that the difference between L and WNT3A CM treated cells at the indicated TRAIL dose is significant with a p-value of <0.01, calculated using Student’s t-test. C) A375 cells were treated with rhTRAIL and WNT3A in the absence and presence of the pan-caspase inhibitor zVAD-FMK (100 µM), and then analyzed for cleaved PARP at 24 hours. C) A375 cells were pre-treated with siRNA specific for β-catenin (<i>CTNNB1</i>) or non-targeting control siRNA for 48 hours. Cells were then treated with rhTRAIL (20 ng/mL) in the presence of WNT3A CM or L CM. Data represents mean percentages of Annexin V-positive cells (+/- SEM) at 24 hours post-treatment as detected by FACS. P-values were calculated using one way ANOVA and Tukey’s post-test analysis. A parallel immunoblot (lower panel) confirms knockdown of β-catenin. The experiments in A and B are representative of at least three independent experiments with similar results.</p

Melanoma cells exhibit diverse apoptotic response to WNT3A plus TRAIL.

<p>Several human melanoma cell lines were assessed for apoptosis at 24 hours upon treatment with rhTRAIL (20 ng/ml) in the absence (10% L CM) and presence of 10% WNT3A CM. Statistically significant increases in Annexin V-positive cells were seen in three cell lines. Data represents the mean percentage of apoptotic cells for three replicates/group (* p<0.05; **p<0.01; ***p<0.001).</p

Depletion of AXIN1 sensitizes melanoma cells to TRAIL-mediated apoptosis.

<p>Melanoma cell lines indicated were pre-treated with control versus <i>AXIN1</i>-specific siRNA for 48 hours. Cells were then treated either WNT3A CM or L CM in addition to rhTRAIL (20ng/mL). The percentage of apoptotic cells was determined by Annexin V binding using FACS. Data represents the mean percentage of apoptotic cells at 24 hours post-treatment +/- SEM. P-values were calculated by one-way ANOVA with a Tukey’s post-test analysis. * indicates p <0.05 compared to either L or WNT3A cells treated with control siRNA.</p

Matrix ChIP analysis of RNA polymerase II (Pol II) <i>Tnf-α</i> genes following unilateral kidney I/R and LPS injection.

<p>Sheared cross-linked renal cortex chromatin from mice were assayed using antibodies to the Pol II N-terminus and CTD modifications. ChIP DNA were analyzed at the Tnf-α first and last exon and CypA first exon in real-time PCR. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input. Schematic of the genes is shown above the graphs; exons are shown as rectangles (taller and shorter rectangles represent translated and untranslated regions), lines represent introns. Black boxes shows location of the amplicon.</p

Synchronous Recruitment of Epigenetic Modifiers to Endotoxin Synergistically Activated Tnf-α Gene in Acute Kidney Injury

<div><p>Background</p><p>As a consequence of acute kidney injury (AKI), proximal tubular cells hyperrespond to endotoxin (lipopolysaccharide, LPS) by exaggerated renal Tnf-α Production. This LPS hyperresponsiveness is transcriptionally mediated. The epigenetic pathways that control these responses are unknown.</p><p>Methods/Findings</p><p>We applied multiplex chromatin immunoprecipitation platform (Matrix ChIP) to explore epigenetic pathways that underlie endotoxin hyperresponsiveness in the setting of preceding unilateral renal ischemia/reperfusion (I/R) in mouse AKI model. Endotoxin exposure after I/R resulted in enhanced transcription, manifested by hyperresponsive recruitment of RNA polymerase II (Pol II) at the Tnf-α gene. At this locus, LPS but not I/R increased levels of Pol II C-terminal domain (CTD) phosho-serine2 &5 and induced dephosphorylation of the transcription-repressive histone H4 phospho-serine-1. In contrast, I/R but not LPS increased the transcription-permissive histone phosphorylation (H3 phospho-serine-10, H3.3 phospho-serine-31) at the Tnf-α gene. In agreement with these observations, I/R but not LPS increased activity of cognate kinases (Erk1/2, Msk1/2 and Aurora A) at the Tnf-α locus. Cross-talk of histone phosphorylation and acetylation synergize to active gene expression. I/R and LPS increased histone acetylation. (H3K9/14Ac, H4K5/8/12/16Ac, H2KA5Ac, H2BK4/7Ac). Levels of some histone acetyltransferases at this gene (PCAF and MOF) were increased by I/R but not by LPS, while others were induced by either I/R or LPS and exhibited endotoxin hyperresponsive patterns (GCN5, CBP and p300). The adaptor protein 14-3-3 couples histone phosphorylation with acetylation, and tethers chromatin modifiers/transcription elongation factors to target genes. Both I/R and LPS increased levels of 14-3-3 and several chromatin/transcription modifiers (BRD4, BRG1, HP-1γ and IKKα) at the Tnf-α gene, all exhibiting endotoxin hyperresponsive recruitment patterns similar to Pol II.</p><p>Conclusions</p><p>Our results suggest that I/R and LPS differentially trigger phosphorylation (Pol II and histone) and acetylation (histone) epigenetic pathways that interact at the Tnf-α gene to generate endotoxin hyperresponse in AKI.</p></div

Wnt/β-catenin activation enhances the expression of the pro-apoptotic proteins BIM and PUMA and diminishes levels of anti-apoptotic MCL1.

<p>A) A375 melanoma cells were treated with rhTRAIL (20 ng/mL) in the presence of WNT3A or L CM. To inhibit caspase activity, cells were also treated with 100µM zVAD-FMK. Immunoblotting analysis was performed with the indicated antibodies. B) A375 cells were pre-treated with β-catenin (<i>CTNNB1</i>) versus control siRNA. 48 hours later, the cells were treated with WNT3A CM or L CM. Immunoblot analysis was then performed with the indicated antibodies. C) Graphs represent the fold-change in protein expression in (B) from digitally-quantified immunoblots +/- SEM (n=5 blots from independent experiments). Each condition was normalized to control cells treated with L CM + control siRNA. D) A375 melanoma cells were treated with WNT3A CM versus L CM in the presence or absence of rhTRAIL (20ng/mL) in the presence of zVAD-FMK (100µM). After 24 hours, RNA was collected for cDNA synthesis and analysis by quantitative real-time PCR (qRT-PCR). Data represents mean relative expression of the indicated gene based on three replicates/group. Significant differences between groups are indicated.</p

Analysis of renal <i>Tnf-α</i> expression following unilateral kidney I/R and LPS injection.

<p>Total RNA from mice renal cortex was used in RT reactions with random hexamers. cDNA was used in real time PCR with gene specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070322#pone.0070322.s004" target="_blank">Table S1</a>). mRNA level of a given gene in each sample was normalized to βactin transcript. Data are represented as mean±SEM, n = 6 mice in each group.</p