Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2-and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.

We constructed an energetic model to determine the optimal focal point current velocity (i.e. microhabitat) for rainbow trout (Oncorhynchus mykiss) and rosyside dace (Clinostomus funduloides) in Coweeta Creek, a fifth—order stream in North Carolina, USA. Energetic costs were evaluated by quantifying the metabolic expenditure associated with swimming at a given velocity. We estimated bebefits by measuring potential energetic gains of feeding at a given velocity. This included estimates of the ability of the fish to capture prey at different current velocities as well as etimates of the frequency and the energy content of drifting prey at various velocities. We derived separate models for small (53—70 mm SL [standard length]) and medium (71—125 mm SL) trout, and medium (47—52 mm SL) and large (53—70 mm SL) dace for all seasons, deriving net energy gain as a function of current velocity. We predicted fishes would occupy velocities at which net energy gain was maximized. Predicted velocities were compared with those utilized by fishes inhabiting Coweeta Creek. Optimal velocities predicted by energetic models ranged from 7.7 to 22.1 cm/s, and closely matched actual velocity use (average deviation = 2.6 cm/s). Prey capture success appeared to be the most important component in the models. Consequently, we constructed models based solely upon aspects of capture success; the average deviation from veolocity use with these models was only 1.8 cm/s. Thus, the ability of dace and trout to capture prey at varying velocities appears to be the dominant factor affecting microhabitat selection in these species.

Background: Pyrin domains (PYDs) mediate the assembly of inflammasome complexes, but PYD interaction modes are not well characterized. Results: Interaction sites were identified on the PYD of the inflammasome adaptor protein, ASC. Conclusion: ASC PYD has multiple binding sites allowing self-association and interaction with binding partners. Significance: Understanding molecular details of inflammasome assembly may lead to development of anti-inflammatory agents.

The structure of FADD has been solved in solution, revealing that the death effector domain (DED) and death domain (DD) are aligned with one another in an orthogonal, tail-to-tail fashion. Mutagenesis of FADD and functional reconstitution with its binding partners define the interaction with the intracellular domain of CD95 and the prodomain of procaspase-8 and reveal a self-association surface necessary to form a productive complex with an activated "death receptor." The identification of a procaspase-specific binding surface on the FADD DED suggests a preferential interaction with one, but not both, of the DEDs of procaspase-8 in a perpendicular arrangement. FADD self-association is mediated by a "hydrophobic patch" in the vicinity of F25 in the DED. The structure of FADD and its functional characterization, therefore, illustrate the architecture of key components in the death-inducing signaling complex.

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Background COVID-19 may be associated with adverse maternal and neonatal outcomes in pregnancy, but there is little controlled data to quantify the magnitude of these risks or to characterize the epidemiology and risk factors. Objective To quantify the associations of COVID-19 with adverse maternal and neonatal outcomes in pregnancy and to characterize the epidemiology and risk factors. Methods We performed a matched case-control study of pregnant patients with confirmed COVID-19 (cases) who delivered between 16 and 41 weeks’ gestation from March 11-June 11, 2020. Uninfected pregnant women (controls) were matched to COVID-19 cases on a 2:1 ratio based on delivery date. Maternal demographic characteristics, COVID-19 symptoms, laboratory evaluations, obstetrical and neonatal outcomes, and clinical management were chart abstracted. The primary outcomes included (i) a composite of adverse maternal outcome, defined as preeclampsia, venous thromboembolism, antepartum admission, maternal intensive care unit admission, need for mechanical ventilation, supplemental oxygen, or maternal death; and (ii) a composite of adverse neonatal outcome, defined as respiratory distress syndrome, intraventricular hemorrhage, necrotizing enterocolitis, five-minute Apgar score <5, persistent category 2 fetal heart rate tracing despite intrauterine resuscitation, or neonatal death. In order to quantify the associations between exposure to mild and severe/critical COVID-19 and adverse maternal and neonatal outcomes, unadjusted and adjusted analyses were performed using conditional logistic regression (to account for matching), with matched-pair odds ratio (OR) and 95% confidence interval (CI) based on 1000 bias-corrected bootstrap resampling as the effect measure. Associations were adjusted for potential confounders. Results 61 confirmed COVID-19 cases were enrolled during the study period (mild disease: n=54, 88.5%; severe disease: n=6, 9.8%; and critical disease: n=1, 1.6%). The odds of adverse composite maternal outcome were 3.4 times higher among cases compared to controls (18.0% versus 8.2%, adjusted OR 3.4, 95% CI 1.2-13.4). The odds of adverse composite neonatal outcome were 1.7 times higher in the case group compared to the control group (18.0% versus 13.9%, adjusted OR 1.7, 95% CI 0.8-4.8). Stratified analyses by disease severity indicated that the morbidity associated with COVID-19 in pregnancy was largely driven by the severe/critical disease phenotype. Major risk factors for associated morbidity were Black and Hispanic race, advanced maternal age, medical comorbidities, and antepartum admissions related to COVID-19. Conclusions COVID-19 during pregnancy is associated with increased risk for adverse maternal and neonatal outcomes, an association that is primarily driven by morbidity associated with severe/critical COVID-19. Black and Hispanic race, obesity, advanced maternal age, medical comorbiditi...

We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus.

PEA-15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA-15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been determined using NMR spectroscopy and its interaction with ERK de®ned by characterization of mutants that modulate ERK function. PEA-15 is composed of an N-terminal death effector domain (DED) and a C-terminal tail of irregular structure. NMR footprinting' and mutagenesis identi®ed elements of both the DED and tail that are required for ERK binding. Comparison of the DED-binding surface for ERK2 with the death domain (DD)-binding surface of Drosophila Tube revealed an unexpected similarity between the interaction modes of the DD and DED motifs in these proteins. Despite a lack of functional or sequence similarity between PEA-15 and Tube, these proteins utilize a common surface of the structurally similar DD and DED to recognize functionally diverse targets. Keywords: death domain/MAP kinase/NMR/ three-dimensional structure IntroductionThe death effector domain (DED) is one of several small protein recognition modules that mediate the assembly of complexes required for signal transduction in programmed cell death. DEDs found in the adaptor protein FADD and the proforms of the initiator caspases, caspase-8 (FLICE, MACH) and caspase-10, play a pivotal role in the initiation of death receptor-mediated apoptosis, whereas DEDs in viral or cellular FLICE-inhibitory proteins (FLIPs) have the ability to block apoptosis (Ashkenazi and Dixit, 1998;Krammer, 2000). The DED, together with the structurally related death domain (DD) and caspase recruitment domain (CARD), are members of the death motif superfamily characterized by a conservedsix a-helix bundle structure (Aravind et al., 1999;Hofmann, 1999;Fesik, 2000). In addition to a common three-dimensional (3D) fold, these protein domains typically associate via homotypic interactions (DD±DD, DED± DED or CARD±CARD) with complementary domains in their binding partners. Surprisingly, no common protein interaction surface has been discernible for these structurally related motifs (Jeong et al., 1999;Qin et al., 1999;Xiao et al., 1999;Fesik, 2000).DED-containing proteins are involved in other cellular signaling events besides the regulation of apoptosis. For example, the phosphoprotein enriched in astrocytes (PEA-15) activates the extracellular signal receptor-activated kinases (ERK1/2), members of the MAP kinase family Formstecher et al., 2001). PEA-15 is a small protein (15 kDa) that was ®rst identi®ed as an abundant phosphoprotein in brain astrocytes (Araujo et al., 1993) and subsequently was shown to be widely expressed in human tissues and highly conserved among mammals (Danziger et al., 1995;Estelle Âs et al., 1996;Ramos et al., 1998). Several studies have shown that PEA-15 regulates multiple cellular functions including Fas-and tumor necrosis factor-a (TNF-a)-induced apopt...