Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases.

Diverse inflammatory conditions, from infections to autoimmune disease, are often associated with cellular damage and death. Apoptotic cell death has evolved to minimize its inflammatory potential. By contrast, necrotic cell death via necroptosis and pyroptosis-driven by membrane-damaging MLKL and gasdermins, respectively-can both initiate and propagate inflammatory responses. In this review, we provide insights into the function and regulation of MLKL and gasdermin necrotic effector proteins and drivers of plasma membrane rupture. We evaluate genetic evidence that MLKL- and gasdermin-driven necrosis may either provide protection against, or contribute to, disease states in a context-dependent manner. These cumulative insights using gene-targeted mice underscore the necessity for future research examining pyroptotic and necroptotic cell death in human tissue, as a basis for developing specific necrotic inhibitors with the potential to benefit a spectrum of pathological conditions.

RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

Oligomerization-driven MLKL ubiquitylation antagonizes necroptosis.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

Molecular dissection of the pseudokinase ZED1 expands effector recognition to the tomato immune receptor ZAR1.

The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a bacterial pathogen recognition hub that mediates resistance by guarding host kinases for modification by pathogen effectors. The pseudokinase HOPZ-ETI DEFICIENT 1 (ZED1) is the only known ZAR1-guarded protein that interacts directly with a pathogen effector, HopZ1a, from the bacterial pathogen Pseudomonas syringae, making it a promising system for rational design of effector recognition for plant immunity. Here, we conducted an in-depth molecular analysis of ZED1. We generated a library of 164 random ZED1 mutants and identified 50 mutants that could not recognize the effector HopZ1a when transiently expressed in Nicotiana benthamiana. Based on our random mutants, we generated a library of 27 point mutants and found evidence of minor functional divergence between Arabidopsis (Arabidopsis thaliana) and N. benthamiana ZAR1 orthologs. We leveraged our point mutant library to identify regions in ZED1 critical for ZAR1 and HopZ1a interactions and identified two likely ZED1-HopZ1a binding conformations. We explored ZED1 nucleotide and cation binding activity and showed that ZED1 is a catalytically dead pseudokinase, functioning solely as an allosteric regulator upon effector recognition. We used our library of ZED1 point mutants to identify the ZED1 activation loop regions as the most likely cause of interspecies ZAR1-ZED1 incompatibility. Finally, we identified a mutation that abolished ZAR1-ZED1 interspecies incompatibility while retaining the ability to mediate HopZ1a recognition, which enabled recognition of HopZ1a through tomato (Solanum lycopersicum) ZAR1. This provides an example of expanded effector recognition through a ZAR1 ortholog from a non-model species.

The Highway to Hell: A RIP Kinase-Directed Shortcut to Inflammatory Cytokine Production.

RIPK1 and RIPK3 are well-known signaling traffic cops in innate immunity. In this issue of Immunity, Degterev and colleagues show that when they blow the whistle on bacterial infection, they quickly point a white-gloved hand down the express route to inflammatory cytokine production.

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death.

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production.

Inhibitors identify an auxiliary role for mTOR signalling in necroptosis execution downstream of MLKL activation.

Necroptosis is a lytic and pro-inflammatory form of programmed cell death executed by the terminal effector, the MLKL (mixed lineage kinase domain-like) pseudokinase. Downstream of death and Toll-like receptor stimulation, MLKL is trafficked to the plasma membrane via the Golgi-, actin- and microtubule-machinery, where activated MLKL accumulates until a critical lytic threshold is exceeded and cell death ensues. Mechanistically, MLKL's lytic function relies on disengagement of the N-terminal membrane-permeabilising four-helix bundle domain from the central autoinhibitory brace helix: a process that can be experimentally mimicked by introducing the R30E MLKL mutation to induce stimulus-independent cell death. Here, we screened a library of 429 kinase inhibitors for their capacity to block R30E MLKL-mediated cell death, to identify co-effectors in the terminal steps of necroptotic signalling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJI308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits mammalian target of rapamycin (mTOR) signalling or regulators thereof, and blocked constitutive cell death executed by R30E MLKL. Our study implicates mTOR signalling as an auxiliary factor in promoting the transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Large-scale genomic investigation of pediatric cholestasis reveals a novel hepatorenal ciliopathy caused by PSKH1 mutations.

PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.

Posttraumatic stress and delay discounting: a meta-analytic review.

Delay discounting-the extent to which individuals show a preference for smaller immediate rewards over larger delayed rewards-has been proposed as a transdiagnostic neurocognitive process across mental health conditions, but its examination in relation to posttraumatic stress disorder (PTSD) is comparatively recent. To assess the aggregated evidence for elevated delay discounting in relation to posttraumatic stress, we conducted a meta-analysis on existing empirical literature. Bibliographic searches identified 209 candidate articles, of which 13 articles with 14 independent effect sizes were eligible for meta-analysis, reflecting a combined sample size of N = 6897. Individual study designs included case-control (e.g. examination of differences in delay discounting between individuals with and without PTSD) and continuous association studies (e.g. relationship between posttraumatic stress symptom severity and delay discounting). In a combined analysis of all studies, the overall relationship was a small but statistically significant positive association between posttraumatic stress and delay discounting (r = .135, p < .0001). The same relationship was statistically significant for continuous association studies (r = .092, p = .027) and case-control designs (r = .179, p < .001). Evidence of publication bias was minimal. The included studies were limited in that many did not concurrently incorporate other psychiatric conditions in the analyses, leaving the specificity of the relationship to posttraumatic stress less clear. Nonetheless, these findings are broadly consistent with previous meta-analyses of delayed reward discounting in relation to other mental health conditions and provide further evidence for the transdiagnostic utility of this construct.

CaMKK2 as an emerging treatment target for bipolar disorder.

Current pharmacological treatments for bipolar disorder are inadequate and based on serendipitously discovered drugs often with limited efficacy, burdensome side-effects, and unclear mechanisms of action. Advances in drug development for the treatment of bipolar disorder remain incremental and have come largely from repurposing drugs used for other psychiatric conditions, a strategy that has failed to find truly revolutionary therapies, as it does not target the mood instability that characterises the condition. The lack of therapeutic innovation in the bipolar disorder field is largely due to a poor understanding of the underlying disease mechanisms and the consequent absence of validated drug targets. A compelling new treatment target is the Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme. CaMKK2 is highly enriched in brain neurons and regulates energy metabolism and neuronal processes that underpin higher order functions such as long-term memory, mood, and other affective functions. Loss-of-function polymorphisms and a rare missense mutation in human CAMKK2 are associated with bipolar disorder, and genetic deletion of Camkk2 in mice causes bipolar-like behaviours similar to those in patients. Furthermore, these behaviours are ameliorated by lithium, which increases CaMKK2 activity. In this review, we discuss multiple convergent lines of evidence that support targeting of CaMKK2 as a new treatment strategy for bipolar disorder.

The Relative Reinforcing Value of Menthol Among Young Adult Cigarette Smokers: Results From a Behavioral Choice Task.

INTRODUCTION: Menthol cigarettes are associated with experimentation and progression to regular use. Although reinforcement processes likely underlie menthol's appeal, the reinforcing value of menthol cigarettes remains unknown. AIMS AND METHODS: This study examined the relative reinforcing value (RRV) of menthol versus nonmenthol cigarettes in young adult menthol (n = 54) and nonmenthol (n = 53) smokers, and differences in menthol's RRV by race, ethnicity, and sexual orientation. Overnight abstinent participants completed a choice task assessing willingness to "work" to click targets on a computer screen to earn menthol or nonmenthol cigarette puffs. A progressive ratio schedule was used where the menthol target had to be clicked progressively more times, over 10 trials, to earn a menthol cigarette puff, while clicks for the nonmenthol target were fixed across trials. RRV for menthol was defined by the breakpoint, or the highest trial (out of to 10) completed for a menthol cigarette puff. Number of clicks for menthol and nonmenthol puffs were also examined. RESULTS: Menthol smokers worked harder for menthol versus nonmenthol cigarette puffs (breakpoint = 9.17; ~1236 clicks vs. 24 clicks). Breakpoint was higher among Hispanic (6.49) versus NH White (4.83) and NH non-White smokers (4.43). In exploratory analyses of interactions of menthol preference with race and ethnicity, nonmenthol Hispanic smokers worked harder for menthol cigarette puffs versus NH non-White and NH White nonmenthol smokers. CONCLUSIONS: Menthol cigarettes are highly reinforcing for young adult menthol and Hispanic smokers. A menthol ban may reduce addiction risk among younger individuals and some minoritized groups of smokers. IMPLICATIONS: This study provides evidence of the greater relative reinforcing value of menthol compared to nonmenthol cigarettes among young adults who had a cigarette flavor preference, suggesting increased addiction risk of menthol cigarettes. Young adult menthol smokers and Hispanic (vs. non-Hispanic) smokers worked harder to earn menthol (vs. nonmenthol) cigarette puffs. Findings add to the evidence base supporting the U.S. Food and Drug Administration's (FDA) intent to ban menthol in cigarettes. Further, prevention messaging campaigns and cessation programs should take into account the reinforcing value of menthol in cigarettes, especially in vulnerable and at-risk populations.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

Horizontal and vertical distribution of Clarireedia spp., in asymptomatic and symptomatic creeping bentgrass cultivars.

Dollar spot is an important disease of both cool- and warm-season turfgrasses caused by six fungal species in the genus Clarireedia, yet the ecology and epidemiology of these pathogens remains poorly understood. The goal of this study was to determine the distribution of Clarireedia in asymptomatic and symptomatic creeping bentgrass (Agrostis stolonifera) in the field using a previously developed qPCR assay. To determine the horizontal distribution of the pathogen, the abundance of Clarireedia spp. was measured in leaf and crown tissue from 90, 1-cm diameter cores spaced 10-cm apart in May 2019 and 2020 (asymptomatic tissue) and August 2019 and July 2020 (symptomatic tissue). Thirty-seven to 69% of cores sampled from asymptomatic turfgrass and 77 to 95% of cores taken from symptomatic turfgrass yielded positive detections for Clarireedia. Spatial analysis indicated that Clarireedia was randomly distributed in the field in both asymptomatic and symptomatic turfgrass. To assess the vertical distribution of the pathogen, the abundance of Clarireedia was measured in the foliar, crown, and thatch layers of 39, 1-cm dia. x 2.5-cm deep cores of creeping bentgrass maintained at fairway height (9.5 mm) during 2019 and 2020. Clarireedia was most abundant in foliar tissue, followed by crown tissue, and thatch (lowest abundance) throughout the two-year study. Both studies provide evidence that Clarireedia is widely distributed in turfgrass swards prior to symptom development and that it can persist within turfgrass as an endophyte. These findings will improve our understanding of Clarireedia epidemiology and may lead to more sustainable dollar spot management.

First Report of Phialocephala bamuru Causing Root Rot on Hard Fescue (Festuca brevipila) in the United States.

Turfgrasses are susceptible to a wide variety of ectotrophic root-infecting (ERI) fungi that cause root rot (Tredway et al., 2023). Among the root rot diseases, fairway patch, caused by Phialocephala bamuru P.T.W. Wong & C. Dong sp. nov., was recently identified and characterized in Australia infecting bermudagrass (Cynodon dactylon) and kikuyu (Pennisetum clandestinum) grass (Wong et al., 2015). Symptoms begin as small, 5-10 cm diameter patches of yellowed turf that may coalesce into larger areas of diseased grass. A characteristic sign of fairway patch is roots colonized by dark brown to black, ectotrophic mycelium. In June 2020, many tan colored, irregular-shaped patches ranging from 10-30 cm in diameter developed on a hard fescue (Festuca brevipila) cultivar 'Beacon' turfgrass field in North Brunswick, New Jersey, USA. The centers of these patches later died and became sunken or filled in partially by recovering hard fescue. The patches grew into tan irregular-shaped rings with diameters up to 3 m by Aug 2023. Symptoms were indicative of a root disease. Five 'Beacon' hard fescue soil cores at the interface of the symptomatic and non-symptomatic area were sampled in Aug 2023. Root and crown samples were observed under a dissecting microscope and dark ectotrophic hyphae were observed on both. Roots with visible ectotrophic mycelium were removed, rinsed in sterile water three times, cut into 5 mm pieces, and plated onto 10% potato dextrose agar amended with streptomycin and gentamicin at 100 mg/L (PDA+). The plates were incubated at 25°C in the dark for 5 days. The most abundant colonies being characteristic long, septate hyphae that were hyaline at the edge and dark brown to black in the center and resembled the fungus described in Wong et al., 2015. These colonies were subcultured onto PDA+ medium and selected for molecular identification. Other less abundant colonies could be identified using morphology after subcultured and had no record being pathogenic to turfgrass. To confirm the isolate's identity, its internal transcribed spacer (ITS) region was amplified in PCR using the ITS1F/ITS4 primers (Bellemain et al., 2010). The amplicon was then sequenced with both ITS1 and ITS4 primers by Sanger sequencing. Sequences were assembled (GenBank #PP000819). The consensus sequence was then BLASTn analyzed with default settings, and the result showed 99.64% sequence identity with P. bamuru (GenBank #MG195534.1). Koch's postulate was conducted in an environmentally controlled growth chamber. Six healthy 'Beacon' hard fescue plugs were sampled from the field. Three of the six plugs (treatment) were each inoculated with P. bamuru by placing 20 g of P. bamuru colonized millets beneath and around the plug before filling the pots with sand. The other three plugs (control) received the same treatment except the P. bamuru colonized millets were autoclaved. The pots were incubated in the growth chamber with a 16 h light period and 25/20°C day/night temperatures. Symptoms resembling those observed in the field appeared on the treatment pots after 21 days of incubation while the control pots remained healthy. The roots from the treatment pots were examined under the dissecting microscope to confirm the colonization of P. bamuru on the roots, and P. bamuru was reisolated and confirmed using the aforementioned morphological traits and molecular assays (GenBank #PP000820). This is the first report of a turfgrass root rot disease caused by P. bamuru in the United States. Further epidemiological, disease ecological, and pathogen biological studies are required to clarify the importance of this disease in the United States and establish proper disease containment or control measures.

Smoking Cessation Tool Utilization Among Individuals with and Without Cancer Across Smoking Status.

Background: There are many tools to assist with cigarette smoking cessation (e.g., counseling, pharmacotherapy). However, tool use among cancer patients is understudied despite the consequences of continued smoking after a cancer diagnosis.Objectives: Study aims included describing and comparing cessation tool use among individuals with and without cancer who were currently smoking and who quit to determine if there are different preferences among those with cancer.Methods: Participants (N = 203) completed an online survey about demographics, cigarette use, and cessation tool use.Results: Eighty-nine participants reported being diagnosed with cancer (45 quit after diagnosis, 44 currently smoking) and 114 reported not having cancer (57 quit, 57 currently smoking). Individuals with cancer who were smoking used more evidence-based resources than those with cancer who quit (B = 1.86, SE = 0.50, p < 0.0001). Individuals with cancer who were smoking used more total cessation resources than participants without cancer who were smoking (B = 2.00, SE = 0.58, p = 0.001), but there was no difference in use of evidence-based resources between these two groups (p > 0.05). Lastly, individuals with cancer who quit also used more total cessation tools (B = 1.23, SE = 0.41, p = 0.003) and evidence-based tools (B = 1.03, SE = 0.36, p = 0.005) than those without cancer who quit.Conclusions: Individuals with cancer may be using more resources before successfully quitting. Cancer patients may need additional help to quit smoking, and further research is needed to better understand unique barriers that preclude quitting among this vulnerable population.

Sleep, Alcohol and Cannabis Use in College Students With and Without Attention-Deficit/Hyperactivity Disorder.

Background: Relations among attention-deficit/hyperactivity disorder (ADHD), sleep, and substance-related negative consequences are largely unknown. In this cross-sectional study, we examined associations among ADHD diagnosis, sleep, and alcohol-related consequences. We also evaluated the independent and interactive effects of sleep and ADHD on alcohol-related negative consequences, above and beyond levels of alcohol use. Methods: College students who drink alcohol with (n = 51) and without (n = 50) ADHD completed an assessment that included a diagnostic interview assessing ADHD, and questionnaire measures of sleep quality, substance use, and associated consequences. Analyses utilized a series of hierarchical linear regression models and explored these aims for cannabis use in a subset of participants (n = 52 participants that used cannabis). Results: College students who drink alcohol with ADHD reported significantly worse sleep quality and more alcohol-related consequences, relative to those without ADHD. When ADHD and sleep quality were included in the model, ADHD-but not sleep quality-was independently associated with alcohol consequences, but not cannabis consequences. There were no moderating effects of ADHD on the associations between sleep and substance-related consequences. Conclusions: Students who drank alcohol with ADHD may be particularly vulnerable to experiencing poor sleep and consequences from their substance use, compared to their heavy drinking peers without ADHD. Future, larger scale studies should consider longitudinal effects as well as underlying mechanisms of risk.

The Structural Basis of Necroptotic Cell Death Signaling.

The recent implication of the cell death pathway, necroptosis, in innate immunity and a range of human pathologies has led to intense interest in the underlying molecular mechanism. Unlike the better-understood apoptosis pathway, necroptosis is a caspase-independent pathway that leads to cell lysis and release of immunogens downstream of death receptor and Toll-like receptor (TLR) ligation. Here we review the role of recent structural studies of the core machinery of the pathway, the protein kinases receptor-interacting protein kinase (RIPK)1 and RIPK3, and the terminal effector, the pseudokinase mixed lineage kinase domain-like protein (MLKL), in shaping our mechanistic understanding of necroptotic signaling. Structural studies have played a key role in establishing models that describe MLKL's transition from a dormant monomer to a killer oligomer and revealing important interspecies differences.