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.

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.

New Approaches to 3D Vision.

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'.

Roles of RIPK3 in necroptosis, cell signaling, and disease.

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.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

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+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

Perceptual decisions and oculomotor responses rely on temporally distinct streams of evidence.

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.

The channel for detecting contrast modulation also responds to density modulation (or vice versa).

In textures composed of black and white dots, we modulated dot density and/or dot contrast in one direction of visual space. Just as Mulligan and MacLeod (Vision Research 28 (1988) 503-519) found a strong reciprocity between density and luminance for dots viewed against a darker background, we found a strong reciprocity between density and contrast: detection thresholds for in-phase modulations of density and contrast were 30% - 55% lower than detection thresholds for density and contrast modulations that were 180° out of phase. These findings support the existence of at least one psychophysical channel that is excited by both density modulations and contrast modulations. A good, quantitative fit to our data can be obtained with a two-channel model.

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.

Reduction in MLKL-mediated endosomal trafficking enhances the TRAIL-DR4/5 signal to increase cancer cell death.

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.

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.

A visual search asymmetry for relative novelty in the visual field based on sensory adaptation.

The ability to detect sudden changes in the environment is important for survival. However, studies of "change blindness" have shown that image differences are hard to detect when a time delay or a mask is imposed between the different images. However, when sensory adaptation is permitted by accurate fixation, we find that change detection is not only possible but asymmetrical: a single changed target amongst 15 unchanging distractors is much easier to detect than a target defined by its lack of change. Although adaptation may selectively reduce the apparent contrast of unchanged objects, the asymmetry in "change salience" cannot be attributed to any such reduction because genuine reductions in target contrast increase, rather than decrease, target detectability. Analogous results preclude attribution to apparent differences between (a) target onset and distractor onset and (b) their temporal frequencies (both flickered at 7.5 Hz, minimizing afterimages). Our results demonstrate a hitherto underappreciated (or unappreciated) advantage conferred by low-level sensory adaptation: it automatically elevates the salience of previously absent objects.

Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition.

While autophagy is thought to be an essential process in some cancer cells, it is unknown if or how such cancer cells can circumvent autophagy inhibition. To address this, we developed a CRISPR/Cas9 assay with dynamic live-cell imaging to measure acute effects of knockout (KO) of autophagy genes compared to known essential and non-essential genes. In some cancer cells, autophagy is as essential for cancer cell growth as mRNA transcription or translation or DNA replication. However, even these highly autophagy-dependent cancer cells evolve to circumvent loss of autophagy by upregulating NRF2, which is necessary and sufficient for autophagy-dependent cells to circumvent ATG7 KO and maintain protein homeostasis. Importantly, however, this adaptation increases susceptibly to proteasome inhibitors. These studies identify a common mechanism of acquired resistance to autophagy inhibition and show that selection to avoid tumor cell dependency on autophagy creates new, potentially actionable cancer cell susceptibilities.

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.

Attention and the motion aftereffect.

We measured the effects of attentional distraction on the time course and asymptote of motion adaptation strength, using visual search performance (percent correct and reaction time). In the first two experiments, participants adapted to a spatial array of moving Gabor patches, either all vertically oriented (Experiment 1) or randomly oriented (Experiment 2). On each trial, the adapting array was followed by a test array in which all of the test patches except one were identical in orientation and movement direction to their retinotopically corresponding adaptors, but the target moved in the opposite direction to its adaptor. Participants were required to identify the location of the changed target with a mouse click. The ability to do so increased with the number of adapting trials. Neither search speed nor accuracy was affected by an attentionally demanding conjunction task at the fixation point during adaptation, suggesting low-level (preattentive) sites in the visual pathway for the adaptation. In Experiment 3, the same participants were required to identify the one element in the test array that was slowly moving. Reaction times in this case were elevated following adaptation, but once again there was no significant effect of the distracting task upon performance. In Experiment 4, participants were required to make eye movements, so that retinotopically corresponding adaptors could be distinguished from spatiotopically corresponding adaptors. Performance in Experiments 1 and 2 correlated positively with reaction times in Experiment 3, suggesting a general trait for adaptation strength.

"Omics" data integration and functional analyses link Enoyl-CoA hydratase, short chain 1 to drug refractory dilated cardiomyopathy.

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 < 2.2e-16). ECHS1:rs10466126 and ECHS1:rs1049951 are in linkage disequilibrium (D' = 1). The interaction (p = 7.84E-08) between ECHS1:rs1049951 and the novel ECHS1:c.41insT variant is associated with drDCM (p < 2.2e-16). The interaction (p = 0.001096) between DBT (Dihydrolipoamide branched chain transacylase E2):384G/S(rs12021720) and ECHS1:rs10466126 is associated with drDCM (p < 2.2e-16). At the mRNA level, there is an association between ECHS1 (log2(foldchange) = 1.63329; q = 0.013927) and DBT (log2(foldchange) = 0.955072; q = 0.0368792) with drDCM. ECHS1 is involved in valine (-log (p = 3.39E00)), isoleucine degradation (p = 0.00457), fatty acid ß-oxidation (-log(p) = 2.83E00), and drug metabolism:cytochrome P450 (z-score = 2.07985196) pathways. The mitochondria (-log(p) = 8.73E00), oxidative phosphorylation (-log(p) = 5.35E00) and TCA-cycle II (-log(p) = 2.70E00) are dysfunctional. CONCLUSIONS: We introduce an integrative data strategy that considers the interplay between the DNA, mRNA, and associated pathways, which represents a possible diagnostic, prognostic, biomarker, and personalized treatment discovery approach in genomically heterogeneous diseases.

Regulation of RIP3 protein stability by PELI1-mediated proteasome-dependent degradation.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a serine-threonine kinase largely essential for necroptotic cell death; it also plays a role in some inflammatory diseases. High levels of RIP3 are likely sufficient to activate necroptotic and inflammatory pathways downstream of RIP3 in the absence of an upstream stimulus. For example, we have previously detected high levels or RIP3 in the skin of Toxic Epidermal Necrolysis patients; this correlates with increased phosphorylation of MLKL found in these patients. We have long surmised that there are molecular mechanisms to prevent anomalous activity of the RIP3 protein, and so prevent undesirable cell death and inflammatory effects when inappropriately activated. Recent discovery that Carboxyl terminus of Hsp 70-Interacting Protein (CHIP) could mediate ubiquitylation- and lysosomedependent RIP3 degradation provides a potential protein that has this capacity. However, while screening for RIP3-binding proteins, we discovered that pellino E3 ubiquitin protein ligase 1 (PELI1) also interacts directly with RIP3 protein; further investigation in this study revealed that PELI1 also targets RIP3 for proteasome-dependent degradation. Interestingly, unlike CHIP, which targets RIP3 more generally, PELI1 preferentially targets kinase active RIP3 that has been phosphorylated on T182, subsequently leading to RIP3 degradation. [BMB Reports 2018; 51(10): 484-485].

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.

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.

Varicella-zoster virus inhibits autophagosome-lysosome fusion and the degradation stage of mTOR-mediated autophagic flux.

Macroautophagy (herein referred to as autophagy) is a lysosomal degradation mechanism that is important for maintaining homeostasis and for coping with cellular stress such as nutrient deprivation. Previously, varicella-zoster virus (VZV) was reported to modulate the autophagy pathway in the host. However, how VZV affects the autophagy pathway is still unclear. In this study, we examined how wild-type rOka and attenuated vOka strains of cell-associated VZV affect autophagy in MRC-5 fibroblasts by using ratiometric flow cytometry and immunoblotting methods. While VZV does not prevent autophagosome formation, we demonstrate that, particularly when autophagy is upregulated, VZV inhibits late-stage autophagic flux, likely at the point where autophagosomes and lysosomes fuse or where vesicle contents are degraded. Importantly, inhibition of autophagy yields higher VZV titers. These results substantially contribute to the current view of the interaction between VZV and autophagy, and to a better understanding of VZV pathogenesis.

An observational study on the sub-acute effects of mephedrone on mood, cognition, sleep and physical problems in regular mephedrone users.

RATIONALE: Mephedrone (4-methylmethcathinone; 4-MMC) is a novel recreational drug similar to methylenedioxymethamphetamine (MDMA) and amphetamine. Several adverse effects have been reported, but little is known about its sub-acute effects. OBJECTIVES: To study sub-acute effects of mephedrone over a period of 9 days. METHODS: Recreational mephedrone users were recruited and followed over a time period of 9 days. It was recorded whether participants consumed mephedrone or not within the period of testing; those who did were compared to those who did not. Forty-six regular mephedrone users (22 males, 24 females) participated, 21 participants voluntarily opted to consume mephedrone 1-3 days after baseline and 25 opted to abstain. Participants were assessed at baseline on a multitude of measures and provided daily reports on cognition, sleep, mood, physical problems, mephedrone cravings and substance use on each subsequent day of the study. The study controlled for psychopathology, sleep, past and current substance use, impulsivity and demographics. RESULTS: Those who consumed mephedrone reported persistent negative mood, physical problems and fatigue, compared to those who did not-after controlling for baseline group differences in sleep and subsequent alcohol and cannabis use. CONCLUSIONS: The results provide the first prospective evidence of the duration and extent of specific undesirable sub-acute effects of mephedrone in regular recreational users and indicate sub-acute effects of mephedrone on mood, fatigue and physical symptoms.