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In this study, a rectangular microstrip feed antenna on a fire-retarded substrate is presented. The antenna is designed using Computer Simulation Tool (CST) software. Different materials are used for the substrate, and the electromagnetic properties of the proposed structure are analyzed for each material. The EM property variation for each material is comparatively presented. The proposed antenna has CSRR (Complementary Split-Ring Resonator) which is engraved on the radiating element, and the shape of the CSRR is the hexagon. The proposed metamaterial-inspired antenna exhibits multiband operation at 2.1 GHz, 2.6 GHz, 4.6 GHz, 5.5 GHz, 6.1 GHz, and 7.1 GHz. With the help of material analysis, the fire-retarded substrate is chosen for the design, its excellent EM properties, and cheap cost. The photolithography-based printed antenna is validated with the help of S11, VSWR, gain, directivity, surface current distribution, and radiation pattern. Simple, compact structure, good gain, stable radiation pattern, and excellent EM properties of the fire-retarded substrates make it suitable for the GHz application.

A pentaband antenna is presented based on the conducting copper material printed on an FR4 substrate for the applications operating in the Gigahertz frequencies. The antenna has a substrate material with a dielectric constant of 4.4. The conducting copper is printed on the FR4 substrate acting as the radiating element and ground. The antenna radiating element has a defected circular structure with a cross stub. The proposed structure is operating at 2.64 GHz, 4.87 GHz, 7.86 GHz, 10.74 GHz, and 13.67 GHz. The antenna is simulated using CST software. The antenna is fabricated and validated with the measurement of return loss. The antenna simulated results like surface current distribution, gain, directivity, and radiation pattern prove that the proposed structure with its compact size is the right candidate for the GHz application.

In this paper, a novel 2 × 2 multiple-input multiple-output (MIMO) antenna array with four patch elements is designed. The proposed antenna is the first dual band, operating at two prominent working frequencies: 24 (24.286-25.111) GHz and 77 GHz (75.348–79.688), of automotive radars. This structure is composed of two antenna modules colocated on a single substrate, whereas each module is made up of a corporate fed planar array of two elements. This attractive feature enables us to utilize the antenna in two different ways; either both modules serve as the transmitting/receiving antenna of a monostatic radar or one module serves as a transmitter and the other one as the receiver of a bistatic radar. Most of the existing autonomous radar applications operating at 24 GHz are going to become obsolete, and all countries have plans of shifting towards the 77 GHz band. Hence, our design is very attractive as it operates with the required performance in both the bands with another added feature of the MIMO structure. The placement of antenna elements is also optimized in terms of inter- and intraelement separation of greater than λ/2 so as to ensure high diversity gain of 9.6 dBi. Moreover, the proposed antenna structure with only two antenna elements is able to achieve a high gain of around 11.8 dBi and 11.3 dBi at the dual operating modes of 24 GHz and 77 GHz, respectively. In addition to the above-mentioned benefits, this design also addresses mutual coupling reduction that is a common problem in MIMO structures by using complementary split ring resonator (CSRR) structures. State-of-the-art comparison with the recent literature shows that the proposed antenna has less number of antenna elements, an adequate gain, an excellent VSWR value, and high isolation.

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma. [ABSTRACT FROM AUTHOR]

Cannabis use is associated with reduced prevalence of obesity and diabetes mellitus (DM) in humans and mouse disease models. Obesity and DM are a well-established independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most prevalent liver disease globally. The effects of cannabis use on NAFLD prevalence in humans remains ill-defined. Our objective is to determine the relationship between cannabis use and the prevalence of NAFLD in humans. We conducted a population-based case-control study of 5,950,391 patients using the 2014 Healthcare Cost and Utilization Project (HCUP), Nationwide Inpatient Survey (NIS) discharge records of patients 18 years and older. After identifying patients with NAFLD (1% of all patients), we next identified three exposure groups: non-cannabis users (98.04%), non-dependent cannabis users (1.74%), and dependent cannabis users (0.22%). We adjusted for potential demographics and patient related confounders and used multivariate logistic regression (SAS 9.4) to determine the odds of developing NAFLD with respects to cannabis use. Our findings revealed that cannabis users (dependent and non-dependent) showed significantly lower NAFLD prevalence compared to non-users (AOR: 0.82[0.76-0.88]; p

OBJECTIVES/SPECIFIC AIMS: To assess the effect of cannabis on impaired judgment and health outcomes among heart failure patients in the emergency room. METHODS/STUDY POPULATION: Patients with heart failure presenting to the emergency room. Cannabis with confounders such as income level, insurance type, tobacco use, and age. Discharged against medical advice to assess impaired judgment. Hospitalization rates, length of stay, and death rate to assess health outcomes. Multivariate logistic regression to access the odds of each of these outcomes from cannabis RESULTS/ANTICIPATED RESULTS: Cannabis is associated with impaired outcome (increase in discharge against medical advice). Cannabis have poorer health outcome in terms of more hospitalizations from the emergency department. Cannabis also have better health outcome in terms of shorter length of stay and death rate among cannabis users Versus nonusers. DISCUSSION/SIGNIFICANCE OF IMPACT: This is crucial to inform health care providers to ensure better counseling of cannabis users. This result should also be considered to interpret other publications that shows better outcomes in patients taking cannabis. Cannabis users might only seem to have a better outcome because they tend to discharge against medical advice and thereby die outside the hospital, etc.

Aims: The management of O2, CO2 and SO2 at bottling and the choice of the closure are two key factors of the shelf life of wine in bottles before bringing them to market. The impact of four screw caps, two synthetic and two technical corks was evaluated on a red wine of Merlot/Tannat. Methods and results: Analytical monitoring (O2, CO2, SO2, aphrometric pressure, L*, a*, b*) was carried out during 538 days of storage at 20 °C. Two sensory analyses at 10 and 17 months completed the study. The wine was bottled with an average total oxygen content of 2 mg/L. The heterogeneity intra and inter procedure was controlled, including for the dissolved carbon dioxide content. Conclusion: Unlike closures with highest OTR, the two technical corks and the two screw caps with Saranex seal, harboring the lowest OTR, matched with the wines exhibiting a low total O2 content at equilibrium (from 4th to 18th month), with more free SO2 and less changed colour. However this OTR gradient (5 to 67 μg/d) observed through the physicochemical analyses was not necessarily confirmed by both sensory analyses performed. Significance and impact of study: This study puts into perspective the impact of OTR closure on sensory characteristics evolution of wine consumed during the first two years, especially when the total oxygen at bottling exceeds 1.5 mg/L.

Proposed by FOULON-SOPAGLY society and INRA, a six years applied research program was developed in France to enable creation of create specific lines to elaborate high quality juice with new adapted grapevine varieties and specialized vineyards, with adapted technology. 448 vine varieties from INRA Vassal first international collection of grapevine species were evaluated to finally select 14: 2 V.vinifera,9 hybrids, and 3 from INRA creation (ref A. Bouquet) by hybridization. These last references, obtained from 4 or 5 back crossing between Muscadinia. rotondifolia.X.V.vinifera, have in particular a high level of resistance against mildew and oidium.These varieties have RUN 1 and RPV1. resistance genes, with several other secondary genes on one chromosome locus. This makes access to organic market easier. Agronomic criteria are studied for this selection. Among the 14 selected grapevine species, some have high level of polyphenols (6 times more than alicante). Others (white and red) have a high aromatic potential. Others have a low sugar content 135 g/l to 150 g/l) and adapted sugar/ acidity ratio. -In the cellar: The absence of fermentation and alcohol requires optimization of the process for juice making. For example by increasing polyphenols extraction and colour stability by using thermo-treatment. Flash release process coupled with enzymatic soaking on grapes at 45°C for example, increases 2 or 3 fold polysaccharide juice content, and increases anthocyanins extraction and hence colour intensity. With this process the draining of grape marc and juice production increase. Pressure technology by on line horizontal decanter is possible. -In juice storehouse. Electro membrane process, (oenodia membrane process) with specific membranes gives possibilities to achieve two objectives on juice: - Decrease pH and increase acidity of juice and colour stabilization, without any additive.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo., (© 2024. The Author(s).)

We evaluate the direct and spillover causal effects of a Hepatitis B (HB) vaccination campaign in French schools on the vaccination adherence of the targeted pupils. Using a regression discontinuity design, we show that this campaign created an exogenous shock on vaccination behavior, increasing the HB vaccination rate for children aged 11 and above. At the same time, we show a drop in the measles, mumps, and rubella (MMR) vaccination rate of the targeted pupils and an increase in the parental belief that measles is a benign disease. We interpret these results as a salience effect: the focus on HB vaccination leads to a decrease in the belief that other vaccines are as important. The effect on MMR vaccination was relatively unexpected and may imply a negative externality. Measles is an extremely contagious disease. If the vaccination rate falls, the disease will spread further, raising the question of the net effect of the HB vaccination campaign on the well-being of the population., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL S132P in biological membranes and MLKL S132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, Mlkl S131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease., (© 2023. Springer Nature Limited.)

Niche-conservative species are especially susceptible to changes in their environment, and detecting the negative effects of new stressors in their habitats is vital for safeguarding of these species. In the Arctic, human disturbance including marine traffic and exploration of resources is increasing rapidly due to climate change-induced reduction of sea ice. Here, we show that the narwhal, Monodon monoceros , is extremely sensitive to human-made noise. Narwhals avoided deep diving (> 350 m) with simultaneous reduction of foraging and increased shallow diving activity as a response to either ship sound alone or ship sound with concurrent seismic airgun pulses. Normal behavior decreased by 50 to 75% at distances where received sound levels were below background noise. Narwhals were equally responsive to both disturbance types, hence demonstrating their acute sensitivity to ship sound. This sensitivity coupled with their special behavioral-ecological strategy including a narrow ecological niche and high site fidelity makes them thus especially vulnerable to human impacts in the Arctic.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma., Competing Interests: Declaration of interests A.L.S. and J.M.M. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl -/- and Ripk3 -/- mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl -/- female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans., (© 2023. The Author(s).)

Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown. To address this question, we used an inducible Cre recombinase system to delete endothelial Casp8 in 6-week-old conditionally gene-targeted mice. Extensive whole body vascular gene targeting was confirmed, yet the dominant phenotype was fatal hemorrhagic lesions exclusively within the small intestine. The emergence of these intestinal lesions was not a maladaptive immune response to endothelial Caspase-8-deficiency, but instead relied upon aberrant Toll-like receptor sensing of microbial commensals and tumor necrosis factor receptor signaling. This lethal phenotype was prevented in compound mutant mice that lacked the necroptotic cell death effector, MLKL. Thus, distinct from its systemic role during embryogenesis, our data show that dysregulated microbial- and death receptor-signaling uniquely culminate in the adult mouse small intestine to unleash MLKL-dependent necroptotic hemorrhage after loss of endothelial Caspase-8. These data support a critical role for Caspase-8 in preserving gut vascular integrity in the face of microbial commensals., (© 2022. The Author(s).)

Recanalization with restored cerebral perfusion is the primary goal of thrombolytic therapy in acute ischemic stroke. The identification of adjunctive therapies that can be safely used to enhance thrombolysis in stroke remains an elusive goal. We report here the development of a mouse in situ carotid artery thrombolysis (iCAT) stroke model involving graded cerebral ischemia to induce unihemispheric infarction after thrombotic occlusion of the common carotid artery (CCA). Electrolytic-induced thrombotic occlusion of the left CCA enabled real-time assessment of recanalization and rethrombosis events after thrombolysis with recombinant tissue-type plasminogen activator (rtPA). Concurrent transient stenosis of the right CCA induced unihemispheric hypoperfusion and infarction in the left middle cerebral artery territory. Real-time assessment of thrombolysis revealed recanalization rates <30% in rtPA-treated animals with high rates of rethrombosis. Addition of the direct thrombin inhibitor argatroban increased recanalization rates to 50% and reduced rethrombosis. Paradoxically, this was associated with increased cerebral ischemia and stroke-related mortality (25%-42%). Serial analysis of carotid and cerebral blood flow showed that coadministration of argatroban with rtPA resulted in a marked increase in carotid artery embolization, leading to distal obstruction of the middle cerebral artery. Real-time imaging of carotid thrombi revealed that adjunctive anticoagulation destabilized platelet-rich thrombi at the vessel wall, leading to dislodgement of large platelet emboli. These studies confirm the benefits of anticoagulants in enhancing thrombolysis and large artery recanalization; however, at high levels of anticoagulation (∼3-fold prolongation of activated partial thromboplastin time), this effect is offset by increased incidence of carotid artery embolization and distal middle cerebral artery occlusion. The iCAT stroke model should provide important new insight into the effects of adjunctive antithrombotic agents on real-time thrombus dynamics during thrombolysis and their correlation with stroke outcomes., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL., (© 2022. The Author(s).)

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing., Competing Interests: J.M.M. and A.L.S. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma., (© 2022 The Authors.)

Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord; however, the primary cell death pathway(s) mediating motor neuron demise remain elusive. We recently established that necroptosis, an inflammatory form of regulated cell death, was dispensable for motor neuron death in a mouse model of ALS, implicating other forms of cell death. Here, we confirm these findings in ALS patients, showing a lack of expression of key necroptotic effector proteins in spinal cords. Rather, we uncover evidence for ferroptosis, a recently discovered iron-dependent form of regulated cell death, in ALS. Depletion of glutathione peroxidase 4 (GPX4), an anti-oxidant enzyme and central repressor of ferroptosis, occurred in post-mortem spinal cords of both sporadic and familial ALS patients. GPX4 depletion was also an early and universal feature of spinal cords and brains of transgenic mutant superoxide dismutase 1 (SOD1 G93A ), TDP-43 and C9orf72 mouse models of ALS. GPX4 depletion and ferroptosis were linked to impaired NRF2 signalling and dysregulation of glutathione synthesis and iron-binding proteins. Novel BAC transgenic mice overexpressing human GPX4 exhibited high GPX4 expression localised to spinal motor neurons. Human GPX4 overexpression in SOD1 G93A mice significantly delayed disease onset, improved locomotor function and prolonged lifespan, which was attributed to attenuated lipid peroxidation and motor neuron preservation. Our study discovers a new role for ferroptosis in mediating motor neuron death in ALS, supporting the use of anti-ferroptotic therapeutic strategies, such as GPX4 pathway induction and upregulation, for ALS treatment., (© 2021. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells., (© 2022. The Author(s).)

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL., (© 2021. The Author(s).)

Anthropogenic activities are increasing in the Arctic, posing a threat to niche-conservative species with high seasonal site fidelity, such as the narwhal Monodon monoceros . In this controlled sound exposure study, six narwhals were live-captured and instrumented with animal-borne tags providing movement and behavioural data, and exposed to concurrent ship noise and airgun pulses. All narwhals reacted to sound exposure with reduced buzzing rates, where the response was dependent on the magnitude of exposure defined as 1/distance to ship. Buzzing rate was halved at 12 km from the ship, and whales ceased foraging at 7-8 km. Effects of exposure could be detected at distances > 40 km from the ship.At only a few kilometres from the ship, the received high-frequency cetacean weighted sound exposure levels were below background noise indicating extreme sensitivity of narwhals towards sound disturbance and demonstrating their ability to detect signals embedded in background noise. The narwhal's reactions to sustained disturbance may have a plethora of consequences both at individual and population levels. The observed reactions of the whales demonstrate their auditory sensitivity but also emphasize, that anthropogenic activities in pristine narwhal habitats needs to be managed carefully if healthy narwhal populations are to be maintained.

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

Chronic inflammation of the large intestine is associated with an increased risk of developing colorectal cancer (CRC), the second most common cause of cancer-related deaths worldwide. Necroptosis has emerged as a form of lytic programmed cell death that, distinct from apoptosis, triggers an inflammatory response. Dysregulation of necroptosis has been linked to multiple chronic inflammatory diseases, including inflammatory bowel disease and cancer. Here, we used murine models of acute colitis, colitis-associated CRC, sporadic CRC, and spontaneous intestinal tumorigenesis to investigate the role of necroptosis in these gastrointestinal pathologies. In the Dextran Sodium Sulfate-induced acute colitis model, in some experiments, mice lacking the terminal necroptosis effector protein, MLKL, or its activator RIPK3, exhibited greater weight loss compared to wild-type mice, consistent with some earlier reports. However, the magnitude of weight loss and accompanying inflammatory pathology upon Mlkl deletion varied substantially between independent repeats. Such variation provides a possible explanation for conflicting literature reports. Furthermore, contrary to earlier reports, we observed that genetic deletion of MLKL had no impact on colon cancer development using several mouse models. Collectively, these data do not support an obligate role for necroptosis in inflammation or cancer within the gastrointestinal tract.

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS., Competing Interests: Declaration of Interests S.L.M. declares consultancy with IFM Therapeutics and Quench Bio and received funding from GlaxoSmithKline. S.L.M. and C.-H.Y. are named inventors on International Patent Application No. PCT/AU2019051201. All other authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is proposed to occur by necroptosis, an inflammatory form of regulated cell death. Prior studies implicated necroptosis in ALS based on accumulation of necroptotic markers in affected tissues of patients and mouse models, and amelioration of disease in mutant superoxide dismutase 1 (SOD1 G93A ) mice with inhibition of the upstream necroptotic mediators, receptor interacting protein kinase 1 (RIPK1), and RIPK3. To definitively address the pathogenic role of necroptosis in ALS, we genetically ablated the critical terminal executioner of necroptosis, mixed lineage kinase domain-like protein (MLKL), in SOD1 G93A mice. Disease onset, progression, and survival were not affected in SOD1 G93A mice lacking MLKL. Motor neuron degeneration and activation of neuroinflammatory cells, astrocytes, and microglia, were independent of MLKL expression in SOD1 G93A mice. While RIPK1 accumulation occurred in spinal cords of SOD1 G93A mice in late stage disease, RIPK3 and MLKL expression levels were not detected in central nervous system tissues from normal or SOD1 G93A mice at any disease stage. These findings demonstrate that necroptosis does not play an important role in motor neuron death in ALS, which may limit the potential of therapeutic targeting of necroptosis in the treatment of neurological disorders.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites., Competing Interests: Competing interest statement: E.J.P., J.M.H., S.E.G., A.L.S., C.F., S.N.Y., P.E.C., and J.M.M. contribute to a project developing necroptosis inhibitors with Anaxis Pharma Pty Ltd. A.K. and S.K. are listed as inventors on issued and pending patents on the monobody technology filed by The University of Chicago (US Patent 9512199 B2 and related pending applications).

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinase-domain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.

Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size., (© 2017 by The American Society of Hematology.)

Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury; however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury. These experiments show that the disulfide-driven process of nucleocytoplasmic coagulation (NCC) is the main form of injury-induced protein aggregation. NCC is mechanistically distinct from amyloidogenesis, but still broadly impairs cell function by promoting the aggregation of hundreds of abundant and essential intracellular proteins. A small proportion of the intracellular proteome resists NCC and is instead released from necrotic cells. Notably, the physicochemical properties of NCC-resistant proteins are contrary to those of NCC-sensitive proteins. These observations challenge the dogma that liberation of constituents during necrosis is anarchic. Rather, inherent physicochemical features including cysteine content, hydrophobicity and intrinsic disorder determine whether a protein is released from necrotic cells. Furthermore, as half of the identified NCC-resistant proteins are known autoantigens, we propose that physicochemical properties that control NCC also affect immune tolerance and other host responses important for the restoration of homeostasis after necrotic injury., (© 2016 The Authors.)

Unlabelled: Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function., Summary: Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes., (© 2016 International Society on Thrombosis and Haemostasis.)

The Open Field (OF) test is one of the most commonly used assays for assessing exploratory behaviour and generalised locomotor activity in rodents. Nevertheless, the vast majority of researchers still rely upon costly commercial systems for recording and analysing OF test results. Consequently, our aim was to design a freely available program for analysing the OF test and to provide an accompanying protocol that was minimally invasive, rapid, unbiased, without the need for specialised equipment or training. Similar to commercial systems, we show that our software-called MouseMove-accurately quantifies numerous parameters of movement including travel distance, speed, turning and curvature. To assess its utility, we used MouseMove to quantify unilateral locomotor deficits in mice following the filament-induced middle cerebral artery occlusion model of acute ischemic stroke. MouseMove can also monitor movement within defined regions-of-interest and is therefore suitable for analysing the Novel Object Recognition test and other field-related cognitive tests. To the best of our knowledge, MouseMove is the first open source software capable of providing qualitative and quantitative information on mouse locomotion in a semi-automated and high-throughput fashion, and hence MouseMove represents a sound alternative to commercial movement analysis systems.

Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells - a critical cell that links the innate and adaptive immune systems. We find that plasmin generated on the surface of necrotic cells enhances their phagocytic removal by human monocyte-derived dendritic cells. Plasmin also promoted phagocytosis of protease-resistant microparticles by diverse mouse dendritic cell sub-types both in vitro and in vivo. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Collectively, our findings support a novel role for plasmin formed on dead cells and other phagocytic targets in maintaining tissue homeostasis by increasing the phagocytic function of dendritic cells while simultaneously decreasing their immunostimulatory capacity consistent with producing an immunosuppressive state.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK--an endogenous cytoprotective cascade.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Neovascularization is a hallmark feature of retinopathy of prematurity and diabetic retinopathy. Type 1 angiotensin receptor blockade reduces neovascularization in experimental retinopathy of prematurity, known as oxygen-induced retinopathy (OIR). We investigated in OIR whether inhibiting aldosterone with the aldosterone synthase inhibitor FAD286 reduced neovascularization as effectively as angiotensin receptor blockade (valsartan). OIR was induced in neonatal Sprague-Dawley rats, and they were treated with FAD286 (30 mg/kg per day), valsartan (10 mg/kg per day), or FAD286+valsartan. The cellular sources of aldosterone synthase, the mineralocorticoid receptor, and 11β-hydroxysteroid dehydrogenase 2 were evaluated in retinal cells involved in neovascularization (primary endothelial cells, pericytes, microglia, ganglion cells, and glia). In OIR, FAD286 reduced neovascularization and neovascular tufts by 89% and 67%, respectively, and normalized the increase in vascular endothelial growth factor mRNA (1.74-fold) and protein (4.74-fold) and was as effective as valsartan and FAD286+valsartan. In retina, aldosterone synthase mRNA was reduced with FAD286 but not valsartan. Aldosterone synthase was detected in microglia, ganglion cells, and glia, whereas mineralocorticoid receptor and 11β-hydroxysteroid dehydrogenase 2 were present in all of the cell types studied. Given the location of aldosterone synthase in microglia and their contribution to retinal inflammation and neovascularization in OIR, the effects of FAD286 on microglial density were studied. The increase in microglial density (ionized calcium binding adaptor protein 1 immunolabeling) in OIR was reduced with all of the treatments. In OIR, FAD286 reduced the increase in mRNA for tumor necrosis factor-α, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and monocyte chemoattractant molecule 1. These findings indicate that aldosterone inhibition may be a potential treatment for retinal neovascularization.

Tissue-type plasminogen activator (tPA) is a major protease of the central nervous system. Most studies to date have used in situ- or gel-based zymographic assays to monitor in vivo changes in neural tPA activity. In this study, we demonstrate that the amidolytic assay can be adapted to accurately detect changes in net tPA activity in mouse brain tissues. Using the amidolytic assay, we examined differences in net tPA activity in the cerebral cortex, sub-cortical structures and cerebellum in wildtype (WT) and tPA(-/-) mice, and in transgenic mice selectively overexpressing tPA in neurons. In addition, we assessed changes in endogenous net tPA activity in WT mice following morphine administration, epileptic seizures, traumatic brain injury and ischaemic stroke-neurological settings in which tPA has a known functional role. Under these conditions, acute and compartment-specific regulation of tPA activity was observed. tPA also participates in various forms of chronic neurodegeneration. Accordingly, we assessed tPA activity levels in mouse models of Alzheimer's disease (AD) and spinocerebellar ataxia type-1 (SCA1). Decreased tPA activity was detected in the cortex and subcortex of AD mice, whereas increased tPA activity was found in the cerebellum of SCA1 mice. These findings extend the existing hypotheses that low tPA activity promotes AD, whereas increased tPA activity contributes to cerebellar degeneration. Collectively, our results exemplify the utility of the amidolytic assay and emphasise tPA as a complex mediator of brain function and dysfunction. On the basis of this evidence, we propose that alterations in tPA activity levels could be used as a biomarker for perturbations in brain homeostasis.

Objective: The goal of this study was to investigate the role of platelets in systemic and cardiac inflammatory responses and the development of postinfarct ventricular complications, as well as the efficacy of antiplatelet interventions., Methods and Results: Using a mouse myocardial infarction (MI) model, we determined platelet accumulation and severity of inflammation within the infarcted myocardium by immunohistochemistry and biochemical assays, analyzed peripheral blood platelet-leukocyte conjugation using flow cytometry, and tested antiplatelet interventions, including thienopyridines and platelet depletion. Platelets accumulated within the infarcted region early post-MI and colocalized with inflammatory cells. MI evoked early increase in circulating platelet-leukocyte conjugation mediated by P-selectin/P-selectin glycoprotein ligand-1. Antiplatelet interventions inhibited platelet-leukocyte conjugation in peripheral blood, inflammatory infiltration, content of matrix metalloproteinases or plasminogen activation, and expression of inflammatory mediators in the infarcted myocardium (all P<0.05) and lowered rupture incidence (P<0.01). Clopidogrel therapy alleviated the extent of chronic ventricular dilatation by serial echocardiography., Conclusions: Platelets play a pivotal role in promoting systemic and cardiac inflammatory responses post-MI. Platelets accumulate within the infarcted myocardium, contributing to regional inflammation, ventricular remodeling, and rupture. Antiplatelet therapy reduces the severity of inflammation and risk of post-MI complications, demonstrating a previously unrecognized protective action.

Tissue-type plasminogen activator (tPA) is an extracellular protease that converts plasminogen into plasmin. For tPA to generate plasmin under biologic conditions, a cofactor must first bring tPA and plasminogen into physical proximity. Fibrin provides this cofactor for tPA-mediated plasmin generation in blood. Despite being naturally devoid of fibrin(ogen), tPA-mediated plasmin formation also occurs in the brain. The fibrin-like cofactor(s) that facilitates plasmin formation in the injured brain has remained unknown. Here we show that protein aggregates formed during neuronal injury provide a macromolecular, nonfibrin cofactor that promotes tPA-mediated plasmin formation and subsequent cell breakdown. The binding of plasminogen and tPA to these protein aggregates occurs via distinct mechanisms. Importantly, nonneuronal cell types also exhibit this cofactor effect upon injury, indicating a general phenomenon. This novel cofactor identified in nonviable cells has ramifications for ischemic stroke where tPA is used clinically and where plasmin activity within the injured brain is unwanted. A means of selectively inhibiting the binding of tPA to nonviable cells while preserving its association with fibrin may be of benefit for the treatment of ischemic stroke.

Tissue-type plasminogen activator (t-PA) has recently been identified as a modulator of neuronal plasticity and can initiate conversion of the pro-form of brain-derived neurotrophic factor (BDNF) into its mature form. BDNF also increases t-PA gene expression implicating t-PA as a downstream effector of BDNF function. Here we demonstrate that BDNF-mediated induction of t-PA mRNA requires an increase in t-PA gene transcription. Reporter constructs harboring 9.5 kb of the human t-PA promoter conferred BDNF-responsiveness in transfected mouse primary cortical neurons. This regulation was recapitulated in HEK 293 cells coexpressing the TrkB neurotrophin receptor. t-PA promoter-deletion analysis revealed the presence of two BDNF-responsive domains, one located between -3.07 and -2.5 kb and the other within the proximal promoter. The upstream region was shown to confer BDNF responsiveness in a TrkB-dependent manner when attached to a heterologous promoter. We also identify homologous regions within the murine and bovine t-PA gene promoters and demonstrate that the equivalent upstream murine sequence functions as a BDNF-responsive enhancer when inserted 5' of the human proximal t-PA promoter. Hence, BDNF-mediated induction of t-PA transcription relies on conserved modular promoter elements including a novel upstream BDNF-responsive domain and the proximal t-PA gene promoter.

For over a decade, tissue-type plasminogen activator (t-PA), a serine protease classically known for its profibrinolytic role in the vasculature, has been implicated in numerous aspects of the synaptic plasticity process. But despite being the most intensively studied protease of the CNS, the mechanisms and molecular mediators behind the action of t-PA on synaptic efficacy remain largely undefined. Rather than examine the role of t-PA in proteolytic remodeling of the synaptic extracellular matrix, this review will focus on the evidence that defines t-PA as a direct modulator of neurotransmission and synaptic plasticity by impacting on glutamatergic and dopaminergic pathways.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.