Cryopreservation of lipid bilayers by LEA proteins from Artemia franciscana and trehalose

© 2016 Elsevier Inc. The capacity of Late Embryogenesis Abundant (LEA) proteins and trehalose to protect liposomes against freezing-induced damage was examined by measuring the leakage of 5(6)-carboxyfluorescein (CF). Liposomes were prepared to simulate the lipid compositions of the inner leaflet of the plasma membrane, outer mitochondrial membrane (OMM), and inner mitochondrial membrane (IMM). Two recombinant LEA proteins belonging to Group 3 (AfrLEA2 and AfrLEA3m) were expressed and purified from embryos of Artemia franciscana. Only OMM-like liposomes were significantly protected by AfrLEA2 and AfrLEA3m against freeze-thaw damage; at the highest protein:lipid mass ratio tested, leakage of CF was 56.3% of control with AfrLEA3m and 29.3% with AfrLEA2. By comparison, trehalose provided protection to all compositional types. The greatest stabilization during freezing occurred when trehalose was present on both sides of the bilayer. When mitochondria isolated from rat liver were freeze-thawed in trehalose solution, the OMM remained intact based on the absence of increased oxygen consumption when cytochrome c was added during oxidative phosphorylation (OXPHOS). Respiratory control ratios (OXPHOS/LEAK) were depressed by only 30% after freeze-thawing in trehalose compared to non-frozen controls, which indicated some retention of OXPHOS capacity by the IMM. Trehalose then was loaded into the matrix (0.24 μmol/mg mitochondrial protein) by transient opening of the permeability transition pore, a procedure optimized for retention of OMM integrity. Surprisingly, respiratory control ratios were not improved after freeze-thawing with external plus matrix trehalose, when compared to external trehalose alone. This result could perhaps be explained by insufficient accumulation of matrix trehalose

Quantification of cellular protein expression and molecular features of group 3 LEA proteins from embryos of Artemia franciscana

Late embryogenesis abundant (LEA) proteins are highly hydrophilic, low complexity proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Here, we report the identification of three new mitochondrial LEA proteins in anhydrobiotic embryos of Artemia franciscana, AfrLEA3m-47, AfrLEA3m-43, and AfrLEA3m-29. These new isoforms are recognized by antibody raised against recombinant AfrLEA3m, the original mitochondrial-targeted LEA protein previously reported from these embryos; mass spectrometry confirms all four proteins share sequence similarity. The corresponding messenger RNA (mRNA) species for the four proteins are readily amplified from total complementary DNA (cDNA) prepared from embryos. cDNA sequences of the four mRNAs are quite similar, but each has a stretch of sequence that is absent in at least one of the others, plus multiple single base pair differences. We conclude that all four mitochondrial LEA proteins are products of independent genes. Each possesses a mitochondrial targeting sequence, and indeed Western blots performed on extracts of isolated mitochondria clearly detect all four isoforms. Based on mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis migration, the cytoplasmic-localized AfrLEA2 exists primarily as a homodimer in A. franciscana. Quantification of protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m-43, and AfrLEA3m-29 as a function of development shows that cellular concentrations are highest in diapause embryos and decrease during development to low levels in desiccation-intolerant nauplius larvae. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins. © 2013 Cell Stress Society International

Real-time forecasts of flood hazard and impact: some UK experiences

Major UK floods over the last decade have motivated significant technological and scientific advances in operational flood forecasting and warning. New joint forecasting centres between the national hydrological and meteorological operating agencies have been formed that issue a daily, national Flood Guidance Statement (FGS) to the emergency response community. The FGS is based on a Flood Risk Matrix approach that is a function of potential impact severity and likelihood. It has driven an increased demand for robust, accurate and timely forecast and alert information on fluvial and surface water flooding along with impact assessments. The Grid-to-Grid (G2G) distributed hydrological model has been employed across Britain at a 1km resolution to support the FGS. Novel methods for linking dynamic gridded estimates of river flow and surface runoff with more detailed offline flood risk maps have been developed to obtain real-time probabilistic forecasts of potential impacts, leading to operational trials. Examples of the national-scale G2G application are provided along with case studies of forecast flood impact from (i) an operational Surface Water Flooding (SWF) trial during the Glasgow 2014 Commonwealth Games, (ii) SWF developments under the Natural Hazards Partnership over England & Wales, and (iii) fluvial applications in Scotland

Challenges during diapause and anhydrobiosis: Mitochondrial bioenergetics and desiccation tolerance

© 2018 International Union of Biochemistry and Molecular Biology In preparation for the onset of environmental challenges like overwintering, food limitation, anoxia, or water stress, many invertebrates and certain killifish enter diapause. Diapause is a developmentally-programed dormancy characterized by suppression of development and metabolism. For embryos of Artemia franciscana (brine shrimp), the metabolic arrest is profound. These gastrula-stage embryos depress oxidative metabolism by ~99% during diapause and survive years of severe desiccation in a state termed anhydrobiosis. Trehalose is the sole fuel source for this developmental stage. Mitochondrial function during diapause is downregulated primarily by restricting substrate supply, as a result of inhibiting key enzymes of carbohydrate metabolism. Because proton conductance across the inner membrane is not decreased during diapause, the inference is that membrane potential must be compromised. In the absence of any intervention, the possibility exists that the F1Fo ATP synthase and the adenine nucleotide translocator may reverse, leading to wholesale hydrolysis of cellular ATP. Studies with anhydrobiotes like A. franciscana are revealing multiple traits useful for improving desiccation tolerance that include the expression and accumulation late embryogenesis abundant (LEA) proteins and trehalose. LEA proteins are intrinsically disordered in aqueous solution but gain secondary structure (predominantly α-helix) as water is removed. These protective agents stabilize biological structures including lipid bilayers and mitochondria during severe water stress. © 2018 IUBMB Life, 70(12):1251–1259, 2018

Liposomes with diverse compositions are protected during desiccation by LEA proteins from Artemia franciscana and trehalose

© 2015 Published by Elsevier B.V. Intracellular accumulation of Late Embryogenesis Abundant (LEA) proteins and the disaccharide trehalose is associated with cellular desiccation tolerance in a number of animal species. Two LEA proteins from anhydrobiotic embryos of the brine shrimp Artemia franciscana were tested for the ability to protect liposomes of various compositions against desiccation-induced damage in the presence and absence of trehalose. Damage was assessed by carboxyfluorescein leakage after drying and rehydration. Further, using a cytoplasmic-localized (AfrLEA2) and a mitochondrial-targeted (AfrLEA3m) LEA protein allowed us to evaluate whether each may preferentially stabilize membranes of a particular lipid composition based on the protein\u27s subcellular location. Both LEA proteins were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane (with cardiolipin), outer mitochondrial membrane, and the inner leaflet of the plasma membrane. Thus liposome stabilization by AfrLEA3m or AfrLEA2 was not dependent on lipid composition, provided physiological amounts of bilayer and non-bilayer-forming lipids were present (liposomes with a non-biological composition of 100% phosphatidylcholine were not protected by either protein). Additive protection by LEA proteins plus trehalose was dependent on the lipid composition of the target membrane. Minimal additional damage occurred to liposomes stored at room temperature in the dried state for one week compared to liposomes rehydrated after 24 h. Consistent with the ability to stabilize lipid bilayers, molecular modeling of the secondary structures for AfrLEA2 and AfrLEA3m revealed bands of charged amino acids similar to other amphipathic proteins that interact directly with membranes

Climate adaptation by crop migration.

Many studies have estimated the adverse effects of climate change on crop yields, however, this literature almost universally assumes a constant geographic distribution of crops in the future. Movement of growing areas to limit exposure to adverse climate conditions has been discussed as a theoretical adaptive response but has not previously been quantified or demonstrated at a global scale. Here, we assess how changes in rainfed crop area have already mediated growing season temperature trends for rainfed maize, wheat, rice, and soybean using spatially-explicit climate and crop area data from 1973 to 2012. Our results suggest that the most damaging impacts of warming on rainfed maize, wheat, and rice have been substantially moderated by the migration of these crops over time and the expansion of irrigation. However, continued migration may incur substantial environmental costs and will depend on socio-economic and political factors in addition to land suitability and climate

The Width of a Solar Coronal Mass Ejection and the Source of the Driving Magnetic Explosion

We show that the strength of the magnetic field in the area covered by the flare arcade following a CME-producing ejective solar eruption can be estimated from the final angular width of the CME in the outer corona and the final angular width of the flare arcade. We assume (1) the flux-rope plasmoid ejected from the flare site becomes the interior of the CME plasmoid, (2) in the outer corona (R greater than 2R(sub Sun)) the CME is roughly a spherical plasmoid with legs shaped like a light bulb, and (3) beyond some height in or below the outer corona the CME plasmoid is in lateral pressure balance with the surrounding magnetic field. The strength of the nearly radial magnetic field in the outer corona is estimated from the radial component of the interplanetary magnetic field measured by Ulysses. We apply this model to three well-observed CMEs that exploded from flare regions of extremely different size and magnetic setting. One of these CMEs is an over-and-out CME that exploded from a laterally far offset compact ejective flare. In each event, the estimated source-region field strength is appropriate for the magnetic setting of the flare. This agreement (1) indicates that CMEs are propelled by the magnetic field of the CME plasmoid pushing against the surrounding magnetic field, (2) supports the magnetic-arch-blowout scenario for over-and-out CMEs, and (3) shows that a CME s final angular width in the outer corona can be estimated from the amount of magnetic flux covered by the source-region flare arcade

New Evidence that CMEs are Self-Propelled Magnetic Bubbles

We briefly describe the "standard model" for the production of coronal mass ejections (CMEs), and our view of how it works. We then summarize pertinent recent results that we have found from SOHO observations of CMEs and the flares at the sources of these magnetic explosions. These results support our interpretation of the standard model: a CME is basically a self-propelled magnetic bubble, a low-beta plasmoitl, that (1) is built and unleashed by the tether-cutting reconnection that builds and heats the coronal flare arcade, (2) can explode from a flare site that is far from centered under the full-blown CME in the outer corona, and (3) drives itself out into the solar wind by pushing on the surrounding coronal magnetic field

LEA Proteins during Water Stress: Not Just for Plants Anymore

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many only assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress