PELI1 Selectively Targets Kinase-Active RIP3 for Ubiquitylation-Dependent Proteasomal Degradation.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.

MicroRNAs modulate the noncanonical transcription factor NF-kappaB pathway by regulating expression of the kinase IKKalpha during macrophage differentiation.

MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

Gain control of saccadic eye movements is probabilistic.

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.

RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment.

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.

Metastatic cells are preferentially vulnerable to lysosomal inhibition.

Molecular alterations that confer phenotypic advantages to tumors can also expose specific therapeutic vulnerabilities. To search for potential treatments that would selectively affect metastatic cells, we examined the sensitivity of lineage-related human bladder cancer cell lines with different lung colonization abilities to chloroquine (CQ) or bafilomycin A1, which are inhibitors of lysosome function and autophagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro to highly metastatic cells compared with their less metastatic counterparts. Genetic inactivation of macroautophagy regulators and lysosomal proteins indicated that this was due to greater reliance on the lysosome but not upon macroautophagy. To identify the mechanism underlying these effects, we generated cells resistant to CQ in vitro. Surprisingly, selection for in vitro CQ resistance was sufficient to alter gene expression patterns such that unsupervised cluster analysis of whole-transcriptome data indicated that selection for CQ resistance alone created tumor cells that were more similar to the poorly metastatic parental cells from which the metastatic cells were derived; importantly, these tumor cells also had diminished metastatic ability in vivo. These effects were mediated in part by differential expression of the transcriptional regulator ID4 (inhibitor of DNA binding 4); depletion of ID4 both promoted in vitro CQ sensitivity and restored lung colonization and metastasis of CQ-resistant cells. These data demonstrate that selection for metastasis ability confers selective vulnerability to lysosomal inhibitors and identify ID4 as a potential biomarker for the use of lysosomal inhibitors to reduce metastasis in patients.

The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors.

The physiological function of the adaptor protein TRADD remains unclear because of the unavailability of a TRADD-deficient animal model. By generating TRADD-deficient mice, we found here that TRADD serves an important function in tumor necrosis factor receptor 1 (TNFR1) signaling by orchestrating the formation of TNFR1 signaling complexes. TRADD was essential for TNFR1 signaling in mouse embryonic fibroblasts but was partially dispensable in macrophages; abundant expression of the adaptor RIP in macrophages may have allowed some transmission of TNFR1 signals in the absence of TRADD. Although morphologically normal, TRADD-deficient mice were resistant to toxicity induced by TNF, lipopolysaccharide and polyinosinic-polycytidylic acid. TRADD was also required for TRIF-dependent Toll-like receptor signaling in mouse embryonic fibroblasts but not macrophages. Our findings definitively establish the biological function of TRADD in TNF signaling.

Models for discriminating image blur from loss of contrast.

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.

Calculation Efficiencies for Mean Numerosity.

Relative numerosity is traditionally studied using texture pairs. Observers must decide which member of each pair has the greater total number of texture elements. In the present experiment, textures were segregated into nonoverlapping "sectors" containing between zero and four elements, and our observers were asked to select the texture containing the greater average number of texture elements (per sector). If observers were more sensitive to total numerosity than average numerosity, their performance (quantified by the just-noticeable Weber fraction) should have been better when the two textures occupied the same number of sectors than when they occupied unequal numbers of sectors. However, we recorded Weber fractions between 11% and 13% for all observers in all conditions. This performance was comparable with an otherwise-ideal observer whose decisions were based on between three and five sectors in each texture. We conjecture that traditional numerosity discriminations are based on similarly small numbers of element clusters.

Axicon Lens for Electrons Using a Magnetic Vortex: The Efficient Generation of a Bessel Beam.

We demonstrate experimentally an efficient electron axicon lens using a magnetic vortex. We show that naturally occurring magnetic vortices with circular magnetic moment distributions in a soft-magnetic thin film create conical phase shifts for fast electrons. Such radially symmetric linear phase ramps are equivalent to ideal light optical axicons. We apply this lens to generate efficient nondiffracting electron Bessel beams, which we observe experimentally in through-focus Lorentz images as well as in propagated off-axis electron holograms. This highlights the potential for using magnetic nanostructures as highly efficient and flexible phase plates for crafting desired electron beam shapes.

RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis.

In a synthetic lethality/viability screen, we identified the serine-threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media, in which autophagy is not critical, but selected for in conditions that increase reliance on basal autophagy. RIP1 represses basal autophagy in part due to its ability to regulate the TFEB transcription factor, which controls the expression of autophagy-related and lysosomal genes. RIP1 activates ERK, which negatively regulates TFEB though phosphorylation of serine 142. Thus, in addition to other pro-death functions, RIP1 regulates cellular sensitivity to pro-death stimuli by modulating basal autophagy.

Labeled lines for image blur and contrast.

It has been suggested that blur and contrast discrimination thresholds are limited by a common stage of contrast energy transduction, and that this explains the characteristic "dipper" functions found for contrast and blur discrimination. To test this conjecture, thresholds for discriminating increments from decrements in sharpness/blur, and similarly for contrast, were measured using the same chessboard stimuli with the Method of Single Stimuli (Experiment 1). Using a generic human contrast sensitivity function (HCSF) to calculate energy, thresholds were significantly lower for blur than for contrast. They could be made more similar only by using an implausibly narrow band-pass version of the HCSF. In separate sessions (Experiment 2), observers also attempted to discriminate between blur and contrast changes when they were randomly interleaved (channel discrimination). Channel discrimination thresholds were similar to those predicted from noisy independent channels, consistent with separate labeled lines for the two channels. Experiment 3 measured subthreshold summation of contrast and blur signals, in either energy-add or energy-subtract modes with a two-alternative forced choice task. Both add and subtract modes lowered thresholds. Experiment 4 measured standard T versus C ("dipper") functions for blur, and compared these with T versus C functions when a contrast cue was added to keep energy constant. The finding of a "dipper" function in the latter case suggests that it does not arise from a common energy transduction stage.

Singularimetry of local phase gradients using vortex lattices and in-line holography.

We have developed a differential form of singularimetry, which utilizes phase vortices or intensity gradient singularities as topological fiducial markers in a structured illumination context. This approach analytically measures phase gradients imparted by refracting specimens, yielding quantitative information that is both local and deterministic. We have quantified our phase gradient experiments to demonstrate that lattices of wave field singularities can be used to detect subtle phase gradients imparted by a spherical specimen and fiber optic cylinders.

Measuring Autophagy in the Context of Cancer.

Autophagy plays multiple roles in the formation and progression of cancer, including both suppressive and promotive roles. It not only impacts cancer cell growth and viability directly but also has a significant role through its effects on the tumor microenvironment. Measurement of autophagy can be confusing and sometimes misleading due to the inherent difficulty of measuring both the formation and turnover of molecules involved in the autophagic process. The LC3 proteins serve as autophagosomal markers and are the basis for most of the assays used for measuring autophagy. Since each of the current assays for autophagy has significant limitations, the use of multiple assays for the analysis of autophagy in most contexts is highly advised. Here we outline three assays that are commonly used to evaluate autophagic flux in cells. These assays include the determination of LC3II formation and LC3II and p62 turnover by use of Western Blotting, quantification of LC3 puncta, and the measurement of autophagic flux using tandem labeled mCherry-GFP-LC3.

Further evidence of the heterogeneous nature of impulsivity.

'Impulsivity' refers to a range of behaviours including preference for immediate reward (temporal-impulsivity) and the tendency to make premature decisions (reflection-impulsivity) and responses (motor-impulsivity). The current study aimed to examine how different behavioural and self-report measurements of impulsivity can be categorised into distinct subtypes. Exploratory factor analysis using full information maximum likelihood was conducted on 10 behavioural and 1 self-report measure of impulsivity. Four factors of impulsivity were indicated, with Factor 1 having a high loading of the Stop Signal Task, which measures motor-impulsivity, factor 2 representing reflection-impulsivity with loadings of the Information Sampling Task and Matching Familiar Figures Task, factor 3 representing the Immediate Memory Task, and finally factor 4 which represents the Delay Discounting Questionnaire and The Monetary Choice Questionnaire, measurements of temporal-impulsivity. These findings indicated that impulsivity is not a unitary construct, and instead represents a series of independent subtypes. There was evidence of a distinct reflection-impulsivity factor, providing the first factor analysis support for this subtype. There was also support for additional factors of motor- and temporal-impulsivity. The present findings indicated that a number of currently accepted tasks cannot be considered as indexing motor- and temporal-impulsivity suggesting that additional characterisations of impulsivity may be required.

Induction of autophagy is essential for monocyte-macrophage differentiation.

Monocytes are programmed to undergo apoptosis in the absence of stimulation. Stimuli that promote monocyte-macrophage differentiation not only cause cellular changes, but also prevent the default apoptosis of monocytes. In the present study, we demonstrate that autophagy is induced when monocytes are triggered to differentiate and that the induction of autophagy is pivotal for the survival and differentiation of monocytes. We also show that inhibition of autophagy results in apoptosis of cells that are engaged in differentiation. We found that the differentiation signal releases Beclin1 from Bcl-2 by activating JNK and blocks Atg5 cleavage, both of which are critical for the induction of autophagy. Preventing autophagy induction hampers differentiation and cytokine production; therefore, autophagy is an important transition from monocyte apoptosis to differentiation.

A common visual metric for approximate number and density.

There is considerable interest in how humans estimate the number of objects in a scene in the context of an extensive literature on how we estimate the density (i.e., spacing) of objects. Here, we show that our sense of number and our sense of density are intertwined. Presented with two patches, observers found it more difficult to spot differences in either density or numerosity when those patches were mismatched in overall size, and their errors were consistent with larger patches appearing both denser and more numerous. We propose that density is estimated using the relative response of mechanisms tuned to low and high spatial frequencies (SFs), because energy at high SFs is largely determined by the number of objects, whereas low SF energy depends more on the area occupied by elements. This measure is biased by overall stimulus size in the same way as human observers, and by estimating number using the same measure scaled by relative stimulus size, we can explain all of our results. This model is a simple, biologically plausible common metric for perceptual number and density.

Monocular and binocular mechanisms detect modulations of dot density and dot contrast.

Strong reciprocity has been demonstrated between (1) spatial modulations of dot density and modulations of dot luminance, and (2) modulations of dot density and modulations of dot contrast, in textures. The latter are much easier to detect when presented in phase with one another than when presented 180° out of phase, although out-of-phase modulations can also be detected given sufficient amplitude. This result supports the existence of two detection mechanisms: one that is excited by both density modulations and contrast modulations (quiescent when those modulations are presented 180° out of phase) and another that is relatively insensitive to either density modulations or contrast modulations (thus remaining stimulated regardless of phase angle). We investigate whether the mechanism responsible for detecting out-of-phase modulations depends on high-level computations (downstream from the confluence of monocular signals) or whether both mechanisms are situated at the monocular level of visual processing. Specifically, density-modulated and/or contrast-modulated stimuli were presented monocularly (i.e., to the same eye) or dichoptically (i.e., to opposite eyes). Out-of-phase modulations of density were much easier to detect when presented dichoptically. A dichoptic advantage was also found for out-of-phase density and contrast modulations. These dichoptic advantages imply conscious access to a mechanism at the monocular level of processing. When density modulations were presented dichoptically, 180° out of phase, detection thresholds were highest. Consequently, a mechanism with binocular input must also contribute to the detection of these modulations. We describe a minimal, image-based model for these results that contains one monocular computation and one binocular computation.

NAMPT-Driven M2 Polarization of Tumor-Associated Macrophages Leads to an Immunosuppressive Microenvironment in Colorectal Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a metabolic enzyme with key roles in inflammation. Previous studies have examined the consequences of its upregulated expression in cancer cells themselves, but studies are limited with respect to its role in the other cells within the tumor microenvironment (TME) during colorectal cancer (CRC) progression. Using single-cell RNA sequencing (scRNA-seq) data, it is founded that NAMPT is highly expressed in SPP1+ tumor-associated macrophages (TAMs), a unique subset of TAMs associated with immunosuppressive activity. A NAMPThigh gene signature in SPP1+ TAMs correlated with worse prognostic outcomes in CRC patients. The effect of Nampt deletion in the myeloid compartment of mice during CRC development is explored. NAMPT deficiency in macrophages resulted in HIF-1α destabilization, leading to reduction in M2-like TAM polarization. NAMPT deficiency caused significant decreases in the efferocytosis activity of macrophages, which enhanced STING signaling and the induction of type I IFN-response genes. Expression of these genes contributed to anti-tumoral immunity via potentiation of cytotoxic T cell activity in the TME. Overall, these findings suggest that NAMPT-initiated TAM-specific genes can be useful in predicting poor CRC patient outcomes; strategies aimed at targeting NAMPT may provide a promising therapeutic approach for building an immunostimulatory TME in CRC progression.

The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling.

TNFR-associated death domain protein (TRADD) is a key effector protein of TNFR1 signaling. However, the role of TRADD in other death receptor (DR) signaling pathways, including DR3, has not been completely characterized. Previous studies using overexpression systems suggested that TRADD is recruited to the DR3 complex in response to the DR3 ligand, TNF-like ligand 1A (TL1A), indicating a possible role in DR3 signaling. Using T cells from TRADD knockout mice, we demonstrate in this study that the response of both CD4(+) and CD8(+) T cells to TL1A is dependent upon the presence of TRADD. TRADD knockout T cells therefore lack the appropriate proliferative response to TL1A. Moreover, in the absence of TRADD, both the stimulation of MAPK signaling and activation of NF-κB in response to TL1A are dramatically reduced. Unsurprisingly, TRADD is required for recruitment of receptor interacting protein 1 and TNFR-associated factor 2 to the DR3 signaling complex and for the ubiquitination of receptor interacting protein 1. Thus, our findings definitively establish an essential role of TRADD in DR3 signaling.

Different subtypes of impulsivity differentiate uncontrolled eating and dietary restraint.

The current study explored the relationship between three subtypes of impulsivity (Reflection Impulsivity, Impulsive Choice, and Impulsive Action) and measures of uncontrolled eating (TFEQ-D) and restraint (TFEQ-R). Eighty women classified as scoring higher or lower on TFEQ-D and TFEQ-R completed the Matching Familiar Figures Test (MFFT20), Delay Discounting Task (DDT), a Go No Go task, Balloon Analogue Risk Task (BART), and the Barrett Impulsivity Scale-11 (BIS-11). To test whether these relationships were affected by enforced controls overeating, half of the participants fasted the night before and ate breakfast in the laboratory before testing and half had no such control. Women scoring higher on the TFEQ-D were significantly more impulsive on the MFFT20 and BIS-11 overall but not on DDT, Go No Go or BART. Women scoring higher on TFEQ-R were significantly less impulsive on the Go No Go task but did not differ on other measures. The eating manipulation modulated responses on the BART and BIS-11 non-planning scale depending on TFEQ-D classification. These results confirm recent data that high scores on TFEQ-D are related to impulsivity, but imply this relates more to Reflection Impulsivity rather than Impulsive Choice or Action. In contrast restrained eating was associated with better inhibitory control. Taken together, these results suggest that subtypes of impulsivity further differentiate uncontrolled eating and restraint, and suggest that a poor ability to reflect on decisions may underlie some aspects of overeating.