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Receptor-interacting protein kinase-3 (RIPK3, or RIP3) is an essential protein in the “programmed” and “regulated” cell death pathway called necroptosis. Necroptosis is activated by the death receptor ligands and pattern recognition receptors of the innate immune system, and the findings of many reports have suggested that necroptosis is highly significant in health and human disease. This significance is largely because necroptosis is distinguished from other modes of cell death, especially apoptosis, in that it is highly proinflammatory given that cell membrane integrity is lost, triggering the activation of the immune system and inflammation. Here, we discuss the roles of RIPK3 in cell signaling, along with its role in necroptosis and various pathways that trigger RIPK3 activation and cell death. Lastly, we consider pathological situations in which RIPK3/necroptosis may play a role.

PDF file 98K, Microscopy data with representative photos and quantification

PDF file - 628K, Human normal liver cells (HL 7702) can undergo TRAIL-induced cell death when sensitized by CHX, but not Rg3

PDF file - 225K, Pretreatment, but not postreatment of cells with Rg3 has a sensitization effect in TRAIL- induced cell death

PDF file 31K, Autophagic flux by flow cytometry in vemurafenib treated cells

PDF file - 765K, Sensitization of Rg3 in TRAIL- induced cell death is dose dependent in HepG2 cells

PDF file 186K, Figures legends for supplementary data figures

PDF file 121K, Incucyte growth analysis and western blot analysis

PDF file - 103K

PDF file - 2112K, Short term Rg3 treatment is not cytotoxic in HepG2 cells

PDF file - 61K, Rg3-mediated, but not thapsigargin-mediated sensitization of TRAIL is CHOP dependent

PDF file 2464K, Timeline of treatments and clinical responses

PDF file 117K,Propidium idodide results and western blot analysis

PDF file 17K, Growth curve with vemurafenib treatments

PDF file - 233K, HL 7702 cells do not express high levels of anti-apoptotic proteins

The TRAIL pathway is a potential therapeutic target for anticancer drugs due to selective cytotoxicity in cancer cells. Despite considerable promise, TRAIL or TRAIL receptor agonists have been used thus far with limited success in multiple clinical trials, in part due to acquired TRAIL resistance during chemotherapeutic treatment. Hepatocellular carcinoma (HCC) is a common solid tumor and the third leading cause of cancer-related death worldwide. Classical chemotherapy is not effective for HCC treatment and targeted therapy is limited to sorafenib. Isolated from Panax ginseng CA Meyer, 20(S)-ginsenoside Rg3 is a steroidal saponin with high pharmacologic activity that has been shown to sensitize cells to some chemotherapeutic agents. We investigated the sensitizing effect of Rg3 on TRAIL-induced cell death in HCC cells. We show Rg3 is capable of promoting TRAIL-induced apoptosis in a number of HCC cell lines, including HepG2, SK-Hep1, Huh-7, and Hep3B, but not in normal HL-7702 hepatocytes, indicating that Rg3 sensitization to TRAIL may be specific to cancer cells. Mechanistically, we found that Rg3 upregulates DR5 expression at the transcriptional level. DR5 upregulation in this case is mediated by C/EBP homology protein (CHOP), an important endoplasmic reticulum stress responsive protein. Furthermore, Rg3 is well tolerated and enhances the therapeutic efficacy of TRAIL in mouse xenograft models, suggesting that chemosensitization also occurs in vivo. Taken together, our study identifies Rg3 as a novel anticancer therapeutic agent and supports the further development of Rg3 as a chemosensitizer in combined therapy with TRAIL. Mol Cancer Ther; 12(3); 274–85. ©2012 AACR.

PDF file - 1806K, Supplementary Figure S1. Hierarchical clustering of breast cancer cell lines transduced with an autophagy-focused shRNA library produced two main clusters. Supplementary Figure S2. ATG5, ATG7 and BECN1 shRNAs decreased protein levels in all cell lines tested. Supplementary Figure S3. Autophagy inhibition induces cell death in some cell lines but not others. MCF7, MDAMB231 and MDAMB468 cells were transduced with a lentivirus expressing shRNAs against ATG7 or BECN1, plated and evaluated for cell death with propidium iodide (red) staining after 48 h. Supplementary Figure S4. Triple negative cell lines have constitutively activated JAK/STAT pathway and autophagy regulates their STAT3 phosphorylation status.

MOV file - 3729K, MDAMB231 cells were transduced with a Non-silencing shRNA and imaged for 48h.

MOV file - 3141K, MCF7 cells were transduced with a Non-silencing shRNA and imaged for 48h.

PDF file - 117K, Supplementary table 1. Changes in shRNAs included in the autophagy library per cell line. Cells were transduced with a shRNA library containing autophagy-focused shRNAs. After transduction, the first sample (Post-transduction, PT) was collected. Growth sample (G) was collected after 15 days in culture. Both samples were done in quintuplicate. Table shows comparison of PT versus G samples. shRNAs were considered to be up (Sign Tag = U), or down-regulated (Sign Tag = D) in the growth sample when ANOVA p-value < 0.05 and fold change absolute value > 2.

MOV file - 2644K, MDAMB231 cells were transduced with an ATG7 shRNA and imaged for 48h.

MOV file - 2592K, MDAMB468 cells were transduced with an ATG7 shRNA and imaged for 48h.

MOV file - 2566K, MCF7 cells were transduced with an ATG7 shRNA and imaged for 48h.

New approaches to 3D vision are enabling new advances in artificial intelligence and autonomous vehicles, a better understanding of how animals navigate the 3D world, and new insights into human perception in virtual and augmented reality. Whilst traditional approaches to 3D vision in computer vision (SLAM: simultaneous localization and mapping), animal navigation (cognitive maps), and human vision (optimal cue integration) start from the assumption that the aim of 3D vision is to provide an accurate 3D model of the world, the new approaches to 3D vision explored in this issue challenge this assumption. Instead, they investigate the possibility that computer vision, animal navigation, and human vision can rely on partial or distorted models or no model at all. This issue also highlights the implications for artificial intelligence, autonomous vehicles, human perception in virtual and augmented reality, and the treatment of visual disorders, all of which are explored by individual articles.This article is part of a discussion meeting issue ‘New approaches to 3D vision’.

Background Necroptosis is emerging as a new target for cancer immunotherapy as it is now recognized as a form of cell death that increases tumor immunogenicity, which would be especially helpful in treating immune-desert tumors. De novo synthesis of inflammatory proteins during necroptosis appears especially important in facilitating increased anti-tumor immune responses. While late-stage transcription mediated by NF-κB during cell death is believed to play a role in this process, it is otherwise unclear what cell signaling events initiate this transactivation of inflammatory genes. Methods We employed tandem-affinity purification linked to mass spectrometry (TAP-MS), in combination with the analysis of RNA-sequencing (RNA-Seq) datasets to identify the Tripartite Motif Protein 28 (TRIM28) as a candidate co-repressor. Comprehensive biochemical and molecular biology techniques were used to characterize the role of TRIM28 in RIPK3 activation-induced transcriptional and immunomodulatory events. The cell composition estimation module was used to evaluate the correlation between RIPK3/TRIM28 levels and CD8+ T cells or dendritic cells (DC) in all TCGA tumors. Results We identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28 on serine 473, inhibiting its chromatin binding activity, thereby contributing to the transactivation of NF-κB and other transcription factors, such as SOX9. This leads to elevated cytokine expression, which then potentiates immunoregulatory processes, such as DC maturation. The expression of RIPK3 has a significant positive association with the tumor-infiltrating immune cells populations in various tumor type, thereby activating anti-cancer responses. Conclusion Our data suggest that RIPK3 activation-dependent derepression of TRIM28 in cancer cells leads to increased immunostimulatory cytokine production in the tumor microenvironment, which then contributes to robust cytotoxic anti-tumor immunity.

The effects of tritium on the fracture toughness properties of Type 304L and Type 21-6-9 stainless steel weldments were measured. Weldments were tritium-charged-and-aged and then tested in order to measure the effect of the increasing decay helium content on toughness. The results were compared to uncharged and hydrogen-charged samples. For unexposed weldments having 8-12 volume percent retained delta ferrite, fracture toughness was higher than base metal toughness. At higher levels of weld ferrite, the fracture toughness decreased to values below that of the base metal. Hydrogen-charged and tritium-charged weldments had lower toughness values than similarly charged base metals and toughness decreased further with increasing weld ferrite content. The effect of decay helium content was inconclusive because of tritium off-gassing losses during handling, storage and testing. Fracture modes were dominated by the dimpled rupture process in unexposed weldments. In hydrogen and tritium-exposed weldments, the fracture modes depended on the weld ferrite content. At high ferrite contents, hydrogen-induced transgranular fracture of the weld ferrite phase was observed.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4

Significance Decades of research have shown that, when measured in laboratory conditions, saccadic eye movements are not only variable, due to noise in sensory-motor pathways, but also inaccurate, displaying systematic biases toward smaller movement amplitudes (hypometria) or toward the mean location of the targets (central tendency). Here, we show that such biases are explained by a probabilistic strategy to find the optimal balance of bias and variance that minimizes the expected costs of saccadic errors. Our findings indicate that the oculomotor system possesses a probabilistic representation of its own sensorimotor uncertainty and uses that representation to adjust the parameters of each saccade., Saccades are rapid eye movements that orient the visual axis toward objects of interest to allow their processing by the central, high-acuity retina. Our ability to collect visual information efficiently relies on saccadic accuracy, which is limited by a combination of uncertainty in the location of the target and motor noise. It has been observed that saccades have a systematic tendency to fall short of their intended targets, and it has been suggested that this bias originates from a cost function that overly penalizes hypermetric errors. Here, we tested this hypothesis by systematically manipulating the positional uncertainty of saccadic targets. We found that increasing uncertainty produced not only a larger spread of the saccadic endpoints but also more hypometric errors and a systematic bias toward the average of target locations in a given block, revealing that prior knowledge was integrated into saccadic planning. Moreover, by examining how variability and bias covaried across conditions, we estimated the asymmetry of the cost function and found that it was related to individual differences in the additional time needed to program secondary saccades for correcting hypermetric errors, relative to hypometric ones. Taken together, these findings reveal that the saccadic system uses a probabilistic-Bayesian control strategy to compensate for uncertainty in a statistically principled way and to minimize the expected cost of saccadic errors.

Fracture tests of uncharged and hydrogen-charged single edge bend specimens of additively manufactured 304 stainless steels are simulated using the cohesive zone modeling (CZM) approach. Two-dimensional plane strain finite element analyses without cohesive elements are conducted to identify the values of cohesive energy. Similar analyses using CZM with the trapezoidal traction-separation laws are then conducted. The best-fit cohesive parameters show the values of cohesive strength for the uncharged specimens are higher than those for the hydrogen-charged ones whereas the value of cohesive energy for the uncharged specimens can be either slightly lower or higher than that for the hydrogen-charged ones.

Additive manufacturing (AM) is being considered as a primary manufacturing process for heat pipes. The Savannah River National Laboratory is researching AM to fabricate a hydrogen isotope separation unit for a thermal cycling absorption process that cycles from cryogenic to moderate temperatures and is a pressure boundary. AM is being explored as a replacement technology to improve heat transfer. Simple test samples with three internal geometries were designed and built from type 316L stainless steel using the powder bed fusion laser process. Nine test article geometries were prepared and subjected to tensile and burst testing and were interrogated using x-ray computed tomography and metallography. The parameters selected for processing the tubes produced a consistent product with acceptable tensile properties and microstructures; however, the process parameters used did not produce full density and minor modifications are needed to achieve full density.

Background Large-scale “omics” datasets have not been leveraged and integrated with functional analyses to discover potential drivers of cardiomyopathy. This study addresses the knowledge gap. Methods We coupled RNA sequence (RNA-Seq) variant detection and transcriptome profiling with pathway analysis to model drug refractory dilated cardiomyopathy (drDCM) using the BaseSpace sequencing hub and Ingenuity Pathway Analysis. We used RNA-Seq case-control datasets (n = 6 cases, n = 4 controls), exome sequence familial DCM datasets (n = 3 Italians, n = 5 Italians, n = 5 Chinese), and controls from the HapMap project (n = 5 Caucasians, and n = 5 Asians) for disease modeling and putative mutation discovery. Variant replication datasets: n = 128 cases and n = 15 controls. Source of datasets: NCBI Sequence Read Archive. Statistics: Pairwise differential expression analyses to determine differentially expressed genes and t-tests to calculate p-values. We adjusted for false discovery rates and reported q-values. We used chi-square tests to assess independence among variables, the Fisher’s Exact Tests and overlap p-values for the pathways and p-scores to rank network. Results Data revealed that ECHS1(enoyl-CoA hydratase, short chain 1(log2(foldchange) = 1.63329) hosts a mirtron, MIR3944 expressed in drDCM (FPKM = 5.2857) and not in controls (FPKM = 0). Has-miR3944-3p is a putative target of BAG1 (BCL2 associated athanogene 1(log2(foldchange) = 1.31978) and has-miR3944-5p of ITGAV (integrin subunit alpha V(log2(foldchange) = 1.46107) and RHOD (ras homolog family member D(log2(foldchange) = 1.28851). There is an association between ECHS1:11 V/A(rs10466126) and drDCM (p = 0.02496). The interaction (p = 2.82E-07) between ECHS1:75 T/I(rs1049951) and ECHS1:rs10466126 is associated with drDCM (p

After several seconds of adaptation to a visual array of randomly oriented Gabor patterns, observers can detect and localise a change in the orientation of one of these Gabors, even when the change is preceded by a blank inter-stimulus interval (Morgan & Solomon, 2019a). Previously, we reported that the ability to detect this changed element was unaffected by distracting observers’ attention away from the adapting stimuli by making them look for rare conjunctions of shape and colour at the central fixation point. That finding is replicated in the current paper, and augmented by a demonstration of the attentionally demanding nature of the conjunction search: it significantly impairs discrimination between adapting arrays in which either many or few items briefly lose contrast. Consequently, we can be certain of adaptation’s immunity to the withdrawal of attention, when assessed objectively (i.e. with a performance-based metric).

Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

Crack extensions in arc-shaped tension specimens of uncharged and tritium-charged-and-decayed conventionally forged (CF) 21-6-9 austenitic stainless steels are simulated by two-dimensional finite element analyses using the cohesive zone modeling (CZM) approach with the smooth trapezoidal traction-separation law. The J integrals at the deviation loads of the arc-shaped tension specimens are taken as the reference cohesive energies and the maximum opening stresses ahead of the initial crack tips in the arc-shaped tension specimens are taken as the reference cohesive strengths. The cohesive strengths and cohesive energies are then adjusted to match the maximum loads of the experimental load-displacement curves of the arc-shaped tension specimens. The computational results showed that the computational load-displacement, load-crack extension, crack extension-displacement, and J-R curves of the uncharged and tritium-charged-and-decayed CF steel specimens are compared well with the experimental data.

Perceptual decisions often require the integration of noisy sensory evidence over time. This process is formalized with sequential sampling models, where evidence is accumulated up to a decision threshold before a choice is made. Although intuition suggests that decision formation must precede the preparation of a motor response (i.e., the action used to communicate the choice), neurophysiological findings have suggested that these two processes might be one and the same. To test this idea, we developed a reverse-correlation protocol in which the visual stimuli that influence decisions can be distinguished from those guiding motor responses. In three experiments, we found that the temporal weighting function of oculomotor responses did not overlap with the relatively early weighting function of stimulus properties having an impact on decision formation. These results support a timeline in which perceptual decisions are formed, at least in part, prior to the preparation of a motor response.

Observers can discriminate between blurry and low-contrast images (Morgan, 2017). Wang and Simoncelli (2004) demonstrated that a code for blur is inherent to the phase relationships between localized pattern detectors of different scales. To test whether human observers actually use local phase coherence when discriminating between image blur and loss of contrast, we compared phase-scrambled chessboards with unscrambled chessboards. Although both stimuli had identical amplitude spectra, local phase coherence was disrupted by phase-scrambling. Human observers were required to concurrently detect and identify (as contrast or blur) image manipulations in the 2 × 2 forced-choice paradigm (Nachmias & Weber, 1975; Watson & Robson, 1981) traditionally considered to be a litmus test for “labelled lines” (i.e. detection mechanisms that can be distinguished on the basis of their preferred stimuli). Phase scrambling reduced some observers’ ability to discriminate between blur and a reduction in contrast. However, none of our observers produced data consistent with Watson and Robson's most stringent test for labeled lines, regardless whether phases were scrambled or not. Models of performance fit significantly better when (a) the blur detector also responded to contrast modulations, (b) the contrast detector also responded to blur modulations, or (c) noise in the two detectors was anticorrelated.