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The routes to cell death are many, and distinguishing which path a particular cell may have taken remains a challenge. Wallach et al. review current understanding of how programmed necrotic cell death contributes to inflammation. Science , this issue p. 10.1126/science.aaf2154 Until recently, programmed cell death was conceived of as a single set of molecular pathways. We now know of several distinct sets of death-inducing mechanisms that lead to differing cell-death processes. In one of them—apoptosis—the dying cell affects others minimally. In contrast, programmed necrotic cell death causes release of immunostimulatory intracellular components after cell-membrane rupture. Defining the in vivo relevance of necrotic death is hampered because the molecules initiating it [such as receptor-interacting protein kinase–1 (RIPK1), RIPK3, or caspase-1] also serve other functions. Proteins that participate in late events in two forms of programmed necrosis [mixed lineage kinase domain–like protein (MLKL) in necroptosis and gasdermin-D in pyroptosis] were recently discovered, bringing us closer to identifying molecules that strictly serve in death mediation, thereby providing probes for better assessing its role in inflammation.

The last 60 years has seen unprecedented groundwater extraction and overdraft as well as development of new technologies for water treatment that together drive the advance in intentional groundwater replenishment known as managed aquifer recharge (MAR). This paper is the first known attempt to quantify the volume of MAR at global scale, and to illustrate the advancement of all the major types of MAR and relate these to research and regulatory advancements. Faced with changing climate and rising intensity of climate extremes, MAR is an increasingly important water management strategy, alongside demand management, to maintain, enhance and secure stressed groundwater systems and to protect and improve water quality. During this time, scientific research—on hydraulic design of facilities, tracer studies, managing clogging, recovery efficiency and water quality changes in aquifers—has underpinned practical improvements in MAR and has had broader benefits in hydrogeology. Recharge wells have greatly accelerated recharge, particularly in urban areas and for mine water management. In recent years, research into governance, operating practices, reliability, economics, risk assessment and public acceptance of MAR has been undertaken. Since the 1960s, implementation of MAR has accelerated at a rate of 5%/year, but is not keeping pace with increasing groundwater extraction. Currently, MAR has reached an estimated 10 km3/year, ~2.4% of groundwater extraction in countries reporting MAR (or ~1.0% of global groundwater extraction). MAR is likely to exceed 10% of global extraction, based on experience where MAR is more advanced, to sustain quantity, reliability and quality of water supplies.

Mitochondria are highly dynamic organelles which can change their shape, via processes termed fission and fusion, in order to adapt to different environmental and developmental contexts. Due to the importance of these processes in maintaining a physiologically healthy pool of mitochondria, aberrant cycles of fission/fusion are often seen in pathological contexts. In this review we will discuss how dysregulated fission and fusion promote tumor progression. We focus on the molecular mechanisms involved in fission and fusion, discussing how altered mitochondrial fission and fusion change tumor cell growth, metabolism, motility, and invasion and, finally how changes to these tumor-cell intrinsic phenotypes directly and indirectly impact tumor progression to metastasis. Although this is an emerging field of investigation, the current consensus is that mitochondrial fission positively influences metastatic potential in a broad variety of tumor types. As mitochondria are now being investigated as vulnerable targets in a variety of cancer types, we underscore the importance of their dynamic nature in potentiating tumor progression.

Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the ‘necrosome’), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.

Caspase-8 joins RIPK at the death Caspase-8 mediates apoptosis induced by 'death receptors' on the cell's surface. At the same time, it is able to prevent receptor interacting protein kinase (RIPK)-dependent necrosis. Without caspase-8, mice die during embryonic development, but why this happens is not clear. Two groups show that this lethality is not caused by the absence of apoptosis, but by the RIPK3-dependent necrosis that is unleashed without caspase-8. Mice lacking both caspase-8 and RIP3 develop into viable, immunocompetent adults, but have a progressive lymphoaccumulative disease similar to that in mice that lack the CD95 death receptor. Oberst et al . also show that caspase-8 forms a proteolytically active complex with FLICE-like inhibitory protein long (FLIPL), and that this complex is required for protection against RIP3-dependent necrosis. Caspase-8 mediates apoptosis induced by death receptors. At the same time, this protease is able to prevent RIP-dependent necrosis. Without caspase-8 mice die during their embryonic development. Two papers now show that lethality is not caused by the absence of apoptosis, but by RIP3-dependent necrosis that is unleashed without caspase-8. Mice that lack both caspase-8 and RIP3 develop into viable, immunocompetent, fertile adult mice, but suffer from a progressive lymphoaccumulative disease similar to mice that lack the death receptor CD95. This paper further shows that caspase-8 forms a proteolytically active complex with FLIP L , and that this complex is required for protection against RIP3-dependent necrosis. Caspase-8 has two opposing biological functions—it promotes cell death by triggering the extrinsic pathway of apoptosis, but also has a survival activity, as it is required for embryonic development 1 , T-lymphocyte activation 2 , and resistance to necrosis induced by tumour necrosis factor-α (TNF-α) and related family ligands 3 , 4 . Here we show that development of caspase-8-deficient mice is completely rescued by ablation of receptor interacting protein kinase-3 (RIPK3). Adult animals lacking both caspase-8 and RIPK3 display a progressive lymphoaccumulative disease resembling that seen with defects in CD95 or CD95-ligand (also known as FAS and FASLG, respectively), and resist the lethal effects of CD95 ligation in vivo . We have found that caspase-8 prevents RIPK3-dependent necrosis without inducing apoptosis by functioning in a proteolytically active complex with FLICE-like inhibitory protein long (FLIP L , also known as CFLAR), and this complex is required for the protective function.

Converging evidence suggests that immune-mediated dysfunction plays an important role in the pathogenesis of frontotemporal dementia (FTD). Although genetic studies have shown that immune-associated loci are associated with increased FTD risk, a systematic investigation of genetic overlap between immune-mediated diseases and the spectrum of FTD-related disorders has not been performed. Using large genome-wide association studies (GWASs) (total n = 192,886 cases and controls) and recently developed tools to quantify genetic overlap/pleiotropy, we systematically identified single nucleotide polymorphisms (SNPs) jointly associated with FTD-related disorders-namely, FTD, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and amyotrophic lateral sclerosis (ALS)-and 1 or more immune-mediated diseases including Crohn disease, ulcerative colitis (UC), rheumatoid arthritis (RA), type 1 diabetes (T1D), celiac disease (CeD), and psoriasis. We found up to 270-fold genetic enrichment between FTD and RA, up to 160-fold genetic enrichment between FTD and UC, up to 180-fold genetic enrichment between FTD and T1D, and up to 175-fold genetic enrichment between FTD and CeD. In contrast, for CBD and PSP, only 1 of the 6 immune-mediated diseases produced genetic enrichment comparable to that seen for FTD, with up to 150-fold genetic enrichment between CBD and CeD and up to 180-fold enrichment between PSP and RA. Further, we found minimal enrichment between ALS and the immune-mediated diseases tested, with the highest levels of enrichment between ALS and RA (up to 20-fold). For FTD, at a conjunction false discovery rate < 0.05 and after excluding SNPs in linkage disequilibrium, we found that 8 of the 15 identified loci mapped to the human leukocyte antigen (HLA) region on Chromosome (Chr) 6. We also found novel candidate FTD susceptibility loci within LRRK2 (leucine rich repeat kinase 2), TBKBP1 (TBK1 binding protein 1), and PGBD5 (piggyBac transposable element derived 5). Functionally, we found that the expression of FTD-immune pleiotropic genes (particularly within the HLA region) is altered in postmortem brain tissue from patients with FTD and is enriched in microglia/macrophages compared to other central nervous system cell types. The main study limitation is that the results represent only clinically diagnosed individuals. Also, given the complex interconnectedness of the HLA region, we were not able to define the specific gene or genes on Chr 6 responsible for our pleiotropic signal. We show immune-mediated genetic enrichment specifically in FTD, particularly within the HLA region. Our genetic results suggest that for a subset of patients, immune dysfunction may contribute to FTD risk. These findings have potential implications for clinical trials targeting immune dysfunction in patients with FTD.

The recognition and clearance of dead cells is a process that must occur efficiently to prevent an autoimmune or inflammatory response. Recently, a process was identified wherein the autophagy machinery is recruited to pathogen-containing phagosomes, termed MAPLC3A (LC3)-associated phagocytosis (LAP), which results in optimal degradation of the phagocytosed cargo. Here, we describe the engagement of LAP upon uptake of apoptotic, necrotic, and RIPK3-dependent necrotic cells by macrophages. This process is dependent on some members of the classical autophagy pathway, including Beclin1, ATG5, and ATG7. In contrast, ULK1, despite being required for autophagy, is dispensable for LAP induced by uptake of microbes or dead cells. LAP is required for efficient degradation of the engulfed corpse, and in the absence of LAP, engulfment of dead cells results in increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines. LAP is triggered by engagement of the TIM4 receptor by either phosphatidylserine (PtdSer)-displaying dead cells or PtdSer-containing liposomes. Therefore, the consequence of phagocytosis of dead cells is strongly affected by those components of the autophagy pathway involved in LAP.

Identifying individuals at risk for developing Alzheimer disease (AD) is of utmost importance. Although genetic studies have identified AD-associated SNPs in APOE and other genes, genetic information has not been integrated into an epidemiological framework for risk prediction. Using genotype data from 17,008 AD cases and 37,154 controls from the International Genomics of Alzheimer's Project (IGAP Stage 1), we identified AD-associated SNPs (at p < 10-5). We then integrated these AD-associated SNPs into a Cox proportional hazard model using genotype data from a subset of 6,409 AD patients and 9,386 older controls from Phase 1 of the Alzheimer's Disease Genetics Consortium (ADGC), providing a polygenic hazard score (PHS) for each participant. By combining population-based incidence rates and the genotype-derived PHS for each individual, we derived estimates of instantaneous risk for developing AD, based on genotype and age, and tested replication in multiple independent cohorts (ADGC Phase 2, National Institute on Aging Alzheimer's Disease Center [NIA ADC], and Alzheimer's Disease Neuroimaging Initiative [ADNI], total n = 20,680). Within the ADGC Phase 1 cohort, individuals in the highest PHS quartile developed AD at a considerably lower age and had the highest yearly AD incidence rate. Among APOE ε3/3 individuals, the PHS modified expected age of AD onset by more than 10 y between the lowest and highest deciles (hazard ratio 3.34, 95% CI 2.62-4.24, p = 1.0 × 10-22). In independent cohorts, the PHS strongly predicted empirical age of AD onset (ADGC Phase 2, r = 0.90, p = 1.1 × 10-26) and longitudinal progression from normal aging to AD (NIA ADC, Cochran-Armitage trend test, p = 1.5 × 10-10), and was associated with neuropathology (NIA ADC, Braak stage of neurofibrillary tangles, p = 3.9 × 10-6, and Consortium to Establish a Registry for Alzheimer's Disease score for neuritic plaques, p = 6.8 × 10-6) and in vivo markers of AD neurodegeneration (ADNI, volume loss within the entorhinal cortex, p = 6.3 × 10-6, and hippocampus, p = 7.9 × 10-5). Additional prospective validation of these results in non-US, non-white, and prospective community-based cohorts is necessary before clinical use. We have developed a PHS for quantifying individual differences in age-specific genetic risk for AD. Within the cohorts studied here, polygenic architecture plays an important role in modifying AD risk beyond APOE. With thorough validation, quantification of inherited genetic variation may prove useful for stratifying AD risk and as an enrichment strategy in therapeutic trials.

The peptide natural product nisin has been used as a food preservative for 6 decades with minimal development of resistance. Nisin contains the unusual amino acids dehydroalanine and dehydrobutyrine, which are posttranslationally installed by class I lanthipeptide dehydratases (LanBs) on a linear peptide substrate through an unusual glutamyl-tRNA–dependent dehydration of Ser and Thr. To date, little is known about how LanBs catalyze the transfer of glutamate from charged tRNAGlu to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids. Here, we describe the synthesis of inert analogs that mimic substrate glutamyl-tRNAGlu and the glutamylated peptide intermediate, and determine the crystal structures of 2 LanBs in complex with each of these compounds. Mutational studies were used to characterize the function of the glutamylation and glutamate elimination active-site residues identified through the structural analysis. These combined studies provide insights into the mechanisms of substrate recognition, glutamylation, and glutamate elimination by LanBs to effect a net dehydration reaction of Ser and Thr.