Correction to: NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

In Fig. 6e, the authors noticed that wrong blots for MITF, MART-1 expression/modulation, and for ß-actin were presented, due to the similarity with experiments shown in Figure 5c. Correct MITF, MART-1, and ß-actin blots were added to the revised Fig. 6 shown in the associated Correction. The meaning of the results shown in Fig.6e, as well as the conclusions of this paper were not affected, and the authors regret for this error. These errors have not been fixed in the original Article.

NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.

Synergistic anti-tumor activity and inhibition of angiogenesis by cotargeting of oncogenic and death receptor pathways in human melanoma.

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244-TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244-TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFß1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.

Neurophysiological aspects of chronic motor cortex stimulation.

Chronic motor cortex stimulation is a treatment option for neuropathic drug-resistant pain and possibly associated movement disorders. Preliminary studies suggest the possibility to treat symptoms of Parkinson disease in selected patients. Recently, MCS has been suggested to enhance motor recovery in patients with poststroke hemiparesis. One or more electrodes are placed extradurally over the motor cortex through a burr hole or a small craniotomy, and then connected to a totally implantable neurostimulator. The accurate positioning of the stimulating electrodes over the motor cortex is the key point of the surgical procedure. Motor cortex identification results from the integration of anatomical, neuroradiological, functional, and neurophysiological data, taking into account the huge population variability. Intraoperative neurophysiological mapping of the motor cortex is of paramount importance, in spite of very sophisticated neuroradiological mathematical reconstructions of the motor area. We discuss and compare the different techniques that are utilized by different authors. Moreover, clinical neurophysiology is also helpful in evaluating the results of this neuromodulation procedure and in hypothesizing the mechanisms that are put in play by MCS.

Patient supplemented epidural analgesia after major abdominal surgery with bupivacaine/fentanyl or ropivacaine/fentanyl.

PURPOSE: To compare analgesic efficacy and occurrence of motor block and other side effects during patient supplemented epidural analgesia (PSEA) with either ropivacaine/fentanyl or bupivacaine/fentanyl mixtures. METHODS: In a prospective, randomized, double-blind study, 32 ASAI-III patients undergoing major abdominal surgery received an epidural catheter at the T8- T10, followed by integrated general epidural anesthesia. Postoperative epidural analgesia was provided using a patient controlled pump with either ropivacaine 0.2%/2 microg x ml(-1) fentanyl (group Ropivacaine, n = 16) or bupivacaine 0.125%/2 microg x ml(-1) fentanyl (group Bupivacaine, n = 16) [background infusion 4-6 ml x hr(-1), 1.5 ml Incremental Doses and 20 min lock out]. Verbal pain rating score, number of incremental doses, consumption of epidural analgesic solution and rescue analgesics, sedation (four-point scale), and pulse oximetry were recorded by a blind observer for 48 hr after surgery. RESULTS: No differences in pain relief, motor block, degree of sedation, pulse oximetry and other side effects were observed between the two groups. The number of incremental doses and the volume of analgesic solution infused epidurally were higher in patients receiving the bupivacaine/fentanyl mixture (10 [0-52] I.D. and 236 [204-340] ml) than in patients receiving the ropivacaine/fentanyl solution (5 [0-50] I.D. and 208 [148-260] ml) (P = 0.03 and P = 0.05, respectively). CONCLUSION: Using a ropivacaine 0.2%/2 microg x ml(-1) fentanyl mixture for patient supplemented epidural analgesia after major abdominal surgery provided similar successful pain relief as bupivacaine 0.125%/2 microg x ml(-1) fentanyl, but patients receiving bupivacaine/fentanyl requested more supplemental.

The visual perception of surface orientation from optical motion.

Observers viewed monocular animations of rotating dihedral angles and were required to indicate their perceived structures by adjusting the magnitude and orientation of a stereoscopic dihedral angle. The motion displays were created by directly manipulating various aspects of the image velocity field, including the mean translation, the horizontal and vertical velocity gradients, and the manner in which these gradients changed over time. The adjusted orientation of each planar facet was decomposed into components of slant and tilt. Although the tilt component was estimated with a high degree of accuracy, the judgments of slant exhibited large systematic errors. The magnitude of perceived slant was determined primarily by the magnitude of the velocity gradient scaled by its direction. The results also indicate that higher order temporal derivatives of the moving elements had little effect on observers' judgments.

Infection with equine herpesvirus 1 (EHV-1) strain HVS25A in pregnant mice.

The abortigenic effects of equine herpesvirus 1 (EHV-1) strain HVS25A, given intranasally, were assessed in pregnant BALB/c, C57BL/6J and Quakenbush mice at day 16 of pregnancy. All EHV-1-infected BALB/c mice showed clinical signs typical of EHV-1-induced disease, together with evidence of abortion. However, although there were fetal and neonatal deaths in some C57BL/6J and Quakenbush litters, the respiratory and systemic effects of EHV-1 infection in the dams were inconsistent. BALB/c dams were then inoculated at day 15 of pregnancy with either EHV-1 or rabbit kidney (RK) cell lysate (controls) and animals were killed at days 1-5 post-inoculation (pi), i.e., before the occurrence of abortions. EHV-1-infected mice showed a significant fall in rectal temperature between days 1 and 2 pi and lost weight during the first 4 days pi, demonstrating a significant mean difference in weight gain from the control group at days 2, 3, 4 and 5 pi. Death in utero was seen in five of 90 fetuses of EHV-1-infected mice, but in no fetuses from RK-inoculated mice. On days 1, 2, 3, 4 and 5 pi, the fetuses from EHV-1-infected dams were significantly smaller than those from RK-inoculated dams. Congestion and necrosis of the middle layer of trophoblast and chorionic necrosis were observed in the placentae from EHV-1-infected dams and assessed by a scoring system. Virus was isolated rarely from the fetuses (1/73), placentae (3/72) and uteri (1/16) of EHV-1-infected dams, and only from those killed on day 1 or 2 pi. This indicates that, as in the horse, abortion caused by EHV-1 infection in mice is not necessarily a consequence of fetal infection but may be due to fetal compromise due to vascular effects on the placenta.

The perception of scale-dependent and scale-independent surface structure from binocular disparity, texture, and shading.

The integration of binocular disparity, shading, and texture was measured for two different aspects of three-dimensional structure: (1) shape index, which is a measure of scale-independent structure, and (2) curvedness, which is a measure of scale-dependent structure. Binocular disparity was found to contribute significantly more to judged shape index than it does to judged curvedness, and shading and texture were both found to contribute more to judged curvedness than to judged shape index. These results demonstrate that different cues do not contribute equally to different aspects of perceived surface structure. This finding suggests that, for the case of linear integration, multiple cues to three-dimensional structure do not combine on the basis of a single type of representation shared by all the 'shape-from-X' processes in the visual system.

The perception of surface curvature from optical motion.

Observers viewed the optical flow field of a rotating quadric surface patch and were required to match its perceived structure by adjusting the shape of a stereoscopically presented surface. In Experiment 1, the flow fields included rigid object rotations and constant flow fields with patterns of image acceleration that had no possible rigid interpretation. In performing their matches, observers had independent control of two parameters that determined the surface shape. One of these, called the shape characteristic, is defined as the ratio of the two principle curvatures and is independent of object size. The other, called curvedness, is defined as the sum of the squared principle curvatures and depends on the size of the object. Adjustments of shape characteristic were almost perfectly accurate for both motion conditions. Adjustments of curvedness, on the other hand, were systematically over-estimated and were not highly correlated with the simulated curvedness of the depicted surface patch. In Experiment 2, the same flow fields were masked with a global pattern of curl, divergence, or shear, which disrupted the first-order spatial derivatives of the image velocity field, while leaving the second-order spatial derivatives invariant. The addition of these masks had only negligible effects on observers' performance. These findings suggest that observers' judgments of three-dimensional surface shape from motion are primarily determined by the second-order spatial derivatives of the instantaneous field of image displacements.

The perception of shape and curvedness from binocular stereopsis and structure from motion.

The integration of binocular stereopsis and kinetic depth was measured for two distinct aspects of 3-D structure: (1) shape index, which is a measure of scale-independent structure, and (2) curvedness, which is a measure of scale-dependent structure. We found that motion contributes significantly more to judged shape index than it does to judged curvedness, and stereo contributes significantly more to judged curvedness than it does to judged shape index. This suggests that the differences in the relative contribution of motion and stereo reported here occurred because these two sources do not equally specify the scale-dependent and scale-independent aspects of surface structure. Furthermore, these results seem to be inconsistent with integration models in which the different visual cues all initially contribute to the same single representation of 3-D structure.

Integration of binocular stereopsis and structure from motion in the discrimination of noisy surfaces.

Integration of stereo and motion information was measured on the basis of observers' ability to discriminate between triangle- and sine-wave corrugated surfaces or sinusoidal surfaces of different spatial frequency. Discrimination performance for the triangle-sine task was consistent with independent processing of motion and stereo, but the spatial frequency discrimination task led to performance superior to that predicted by an independent combination of motion and stereo signals, indicating that the integration of stereo and motion depends on the type of 3-dimensional structure observers are required to discriminate. This pattern of results is consistent with the existence of multiple stereoscopic mechanisms suggested by psychophysical and neurophysiological data.

The visual perception of rigid motion from constant flow fields.

Four experiments investigated observers' judgments of rigidity for different types of optical motion. The depicted structural deformations were of two types: (1) those with nonparallel image trajectories that are detectable from the first-order spatiotemporal relations between pairs of views; and (2) those with parallel image trajectories that can only be detected from higher order relations among three or more views. Patterns were composed of smooth flow fields in Experiments 1 and 3, and of wire frame figures in Experiments 2 and 4. For both types of display, the nonrigidity detectable from the first-order spatiotemporal structure of the motion sequence was much more salient than the deformation detectable only from the higher order spatiotemporal structure. These results indicate that observers' judgments of rigidity are based primarily on a two-view analysis, but that some useful information can be obtained under appropriate circumstances from higher order spatiotemporal relations among three or more views.

The visual perception of three-dimensional length.

A set of 4 experiments evaluated observers' sensitivity to three-dimensional (3-D) length, using both discrimination and adjustment paradigms with computer-generated optical patterns and real objects viewed directly in a natural environment. Although observers were highly sensitive to small differences in two-dimensional length for line segments presented in the frontoparallel plane, their discrimination thresholds increased by an order of magnitude when the line segments were presented at random orientations in 3-D space. There were also large failures of constancy, such that the perception of 3-D length varied systematically with viewing distance, even under full-cue conditions.

Systematic distortion of perceived three-dimensional structure from motion and binocular stereopsis.

The geometric relation between physical and perceived space as specified by binocular stereopsis and structure from motion was investigated. Four experimental tasks were used, each of which required a different aspect of three-dimensional (3-D) structure to be performed accurately. To examine whether the transformation between physical and perceptual space preserved the 3-D structural properties required to perform each of our tasks, the constancy of judged shape over changes in a depicted object's viewing distance or orientation was examined. Our results reveal that observers' judgments of 3-D shape from binocular stereopsis and motion contained systematic distortions: Perceived 3-D shape from motion was not invariant over orientation change and perceived 3-D structure from stereo, and motion and stereo in combination was not invariant over changes in viewing distance.