Interplay of buried histidine protonation and protein stability in prion misfolding

Misofolding of mammalian prion proteins (PrP) is believed to be the cause of a group of rare and fatal neurodegenerative diseases. Despite intense scrutiny however, the mechanism of the misfolding reaction remains unclear. We perform nuclear Magnetic Resonance and thermodynamic stability measurements on the C-terminal domains (residues 90–231) of two PrP variants exhibiting different pH-induced susceptibilities to aggregation: the susceptible hamster prion (GHaPrP) and its less susceptible rabbit homolog (RaPrP). The pKa of histidines in these domains are determined from titration experiments, and proton-exchange rates are measured at pH 5 and pH 7. A single buried highly conserved histidine, H187/H186 in GHaPrP/RaPrP, exhibited a markedly down shifted pKa ~5 for both proteins. However, noticeably larger pH-induced shifts in exchange rates occur for GHaPrP versus RaPrP. Analysis of the data indicates that protonation of the buried histidine destabilizes both PrP variants, but produces a more drastic effect in the less stable GHaPrP. This interpretation is supported by urea denaturation experiments performed on both PrP variants at neutral and low pH, and correlates with the difference in disease susceptibility of the two species, as expected from the documented linkage between destabilization of the folded state and formation of misfolded and aggregated species

Human influence on climate in the 2014 southern England winter floods and their impacts

A succession of storms reaching Southern England in the winter of 2013/2014 caused severe floods and £451 million insured losses. In a large ensemble of climate model simulations, we find that, as well as increasing the amount of moisture the atmosphere can hold, anthropogenic warming caused a small but significant increase in the number of January days with westerly flow, both of which increased extreme precipitation. Hydrological modelling indicates this increased extreme 30-day-average Thames river flows, and slightly increased daily peak flows, consistent with the understanding of the catchment’s sensitivity to longer-duration precipitation and changes in the role of snowmelt. Consequently, flood risk mapping shows a small increase in properties in the Thames catchment potentially at risk of riverine flooding, with a substantial range of uncertainty, demonstrating the importance of explicit modelling of impacts and relatively subtle changes in weather-related risks when quantifying present-day effects of human influence on climate

Characterising loss and damage from climate change

Policy-makers are creating mechanisms to help developing countries cope with loss and damage from climate change, but the negotiations are largely neglecting scientific questions about what the impacts of climate change actually are. Mitigation efforts have failed to prevent the continued increase of anthropogenic greenhouse gas (GHG) emissions. Adaptation is now unlikely to be sufficient to prevent negative impacts from current and future climate change1. In this context, vulnerable nations argue that existing frameworks to promote mitigation and adaptation are inadequate, and have called for a third international mechanism to deal with residual climate change impacts, or “loss and damage”2. In 2013, the United Nations Framework Convention on Climate Change (UNFCCC) responded to these calls and established the Warsaw International Mechanism (WIM) to address loss and damage from the impacts of climate change in developing countries3. An interim Executive Committee of party representatives has been set up, and is currently drafting a two-year workplan comprising meetings, reports, and expert groups; and aiming to enhance knowledge and understanding of loss and damage, strengthen dialogue among stakeholders, and promote enhanced action and support. Issues identified as priorities for the WIM thus far include: how to deal with non-economic losses, such as loss of life, livelihood, and cultural heritage; and linkages between loss and damage and patterns of migration and displacement2. In all this, one fundamental issue still demands our attention: which losses and damages are relevant to the WIM? What counts as loss and damage from climate change

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas

Post-antibiotic effect of orbifloxacin against Escherichia coli and Pseudomonas aeruginosa isolates from dogs

Orbifloxacin is a fluoroquinolone drug used widely in companion animal medicine. In this study, we firstly determined post-antibiotic effects (PAEs) and post-antibiotic sub-minimum inhibitory concentrations (MIC) effects (PA-SMEs) of orbifloxacin for two strains each of Escherichia coli and Pseudomonas aeruginosa from dogs, and these parameters were compared with those of enrofloxacin. At twice the MIC, the PAEs of orbifloxacin ranged from -0.28-0.93 h (mean, 0.29 h) for E. coli and -0.18-1.18 h (mean, 0.37 h) for P. aeruginosa. These parameters were not significantly different for E. coli and shorter for P. aeruginosa, compared to enrofloxacin (P < 0.05). Continued exposure to 0.1, 0.2, and 0.3 the MIC of orbifloxacin resulted in average PA-SMEs of 0.55, 1.11, and 2.03 h, respectively, for E. coli, and 1.04, 1.40, and 2.47 h, respectively, for P. aeruginosa. These PA-SMEs, which had no significant differences with those of enrofloxacin, were significantly longer than the corresponding PAEs (P < 0.05). These results suggest that the PA-SME of orbifloxacin for E. coli and P. aeruginosa can be meaningfully prolonged by increase of sub-MICs

Using a game to engage stakeholders in extreme event attribution science

The impacts of weather and climate-related disasters are increasing, and climate change can exacerbate many disasters. Effectively communicating climate risk and integrating science into policy requires scientists and stakeholders to work together. But dialogue between scientists and policymakers can be challenging given the inherently multidimensional nature of the issues at stake when managing climate risks. Building on the growing use of serious games to create dialogue between stakeholders, we present a new game for policymakers called Climate Attribution Under Loss and Damage: Risking, Observing,co-Negotiating (CAULDRON). CAULDRON aims to communicate understanding of the science attributing extreme events to climate change in a memorable and compelling way, and create space for dialogue around policy decisions addressing changing risks and loss and damage from climate change. We describe the process of developing CAULDRON, and draw on observations of players and their feedback to demonstrate its potential to facilitate the interpretation of probabilistic climate information and the understanding of its relevance to informing policy. Scientists looking to engage with stakeholders can learn valuable lessons in adopting similar innovative approaches. The suitability of games depends on the policy context but, if used appropriately, experiential learning can drive co-produced understanding and meaningful dialogue

Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.Harvard University. Society of FellowsNational Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170)United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445)United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.)Wyss Institute of Biologically Inspired EngineeringNational Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01))European School of Molecular Medicine (predoctoral fellowship

Hypernovae and Other Black-Hole-Forming Supernovae

During the last few years, a number of exceptional core-collapse supernovae (SNe) have been discovered. Their kinetic energy of the explosions are larger by more than an order of magnitude than the typical values for this type of SNe, so that these SNe have been called `Hypernovae'. We first describe how the basic properties of hypernovae can be derived from observations and modeling. These hypernovae seem to come from rather massive stars, thus forming black holes. On the other hand, there are some examples of massive SNe with only a small kinetic energy. We suggest that stars with non-rotating black holes are likely to collapse "quietly" ejecting a small amount of heavy elements (Faint supernovae). In contrast, stars with rotating black holes are likely to give rise to very energetic supernovae (Hypernovae). We present distinct nucleosynthesis features of these two types of "black-hole-forming" supernovae. Hypernova nucleosynthesis is characterized by larger abundance ratios (Zn,Co,V,Ti)/Fe and smaller (Mn,Cr)/Fe. Nucleosynthesis in Faint supernovae is characterized by a large amount of fall-back. We show that the abundance pattern of the most Fe deficient star, HE0107-5240, and other extremely metal-poor carbon-rich stars are in good accord with those of black-hole-forming supernovae, but not pair-instability supernovae. This suggests that black-hole-forming supernovae made important contributions to the early Galactic (and cosmic) chemical evolution.Comment: 49 pages, to be published in "Stellar Collapse" (Astrophysics and Space Science; Kluwer) ed. C. L. Fryer (2003

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation