Two‐dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2‐D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue‐native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2‐D native systems are useful means to identify the constituent proteins and their copy numbers in detergent‐labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.     

Non‐native trout limit native brook trout access to space and thermal refugia in a restored large‐river system

We used direct observation via snorkeling surveys to quantify microhabitat use by native brook (Salvelinus fontinalis) and non‐native brown (Salmo trutta) and rainbow (Onchorynchus mykiss) trout occupying natural and restored pool habitats within a large, high‐elevation Appalachian river, United States. Permutational multivariate analysis of variance (PERMANOVA) and subsequent two‐way analysis of variance (ANOVA) indicated a significant difference in microhabitat use by brook and non‐native trout within restored pools. We also detected a significant difference in microhabitat use by brook trout occupying pools in allopatry versus those occupying pools in sympatry with non‐native trout—a pattern that appears to be modulated by size. Smaller brook trout often occupied pools in the absence of non‐native species, where they used shallower and faster focal habitats. Larger brook trout occupied pools with, and utilized similar focal habitats (i.e. deeper, slower velocity) as, non‐native trout. Non‐native trout consistently occupied more thermally suitable microhabitats closer to cover as compared to brook trout, including the use of thermal refugia (i.e. ambient–focal temperature >2°C). These results suggest that non‐native trout influence brook trout use of restored habitats by: (1) displacing smaller brook trout from restored pools, and (2) displacing small and large brook trout from optimal microhabitats (cooler, deeper, and lower velocity). Consequently, benefits of habitat restoration in large rivers may only be fully realized by brook trout in the absence of non‐native species. Future research within this and other large river systems should characterize brook trout response to stream restoration following removal of non‐native species.     

Semi‐synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide‐α‐thioester

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three‐dimensional structure domain was constructed from three segments murine PrP (mPrP)(90–177), mPrP(178–212), and mPrP(213–230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C‐terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Detecting impacts of non‐native species on associated invertebrate assemblages depends on microhabitat

Invasive plants that displace native floral communities can cause changes to associated invertebrate species assemblages. Using a mini‐review of the literature and our own data we add to the still considerable debate about the most effective methods for testing community‐level impacts by invasive species. In endangered saltmarshes of southeast Australia, the non‐native rush Juncus acutus L. is displacing its native congener J. kraussii Hochst., with concurrent changes to floral and faunal assemblages. In two coastal saltmarshes, we tested the hypothesis that the ability to detect differences in the invertebrate assemblage associated with these congeneric rushes depends on the microhabitat of the plant sampled. We used three sampling methods, each targeting specific microhabitats: sweep netting of the plant stems, vacuum sampling of the plant tussock, and vacuum sampling of the ground directly below the plants. Over 3800 individuals and 92 morphospecies were collected across four main taxa: gastropods, crustaceans, hexapods and arachnids. Detection of differences in invertebrate density, richness and composition associated with native compared with non‐native rushes was dependent on the microhabitat sampled and these differences were spatially variable. For example, at one saltmarsh the stems and tussock of J. acutus had a lower density and richness of total invertebrates and hexapods than those of the native J. kraussii. In contrast, crustaceans on the ground were in greater abundance below J. acutus than J. kraussii. This study demonstrates that on occasions where overall differences in the assemblage are not detected between species, differences may become apparent when targeting different microhabitats of the plant. In addition, separately targeting multiple microhabitats likely leads to a greater probability of detecting impacts of invasion. Comparing the invertebrate assemblage without differentiating between or sampling an array of microhabitats can fail to determine the impact of invasive species. These results highlight that a combination of methods targeting different microhabitats is important for detecting differences within the invertebrate community, even for phylogenetically related species.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Co‐varying impacts of land use and non‐native brown trout on fish communities in small streams

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First record of non‐native hybrid catfish Pseudoplatystoma reticulatum × Leiarius marmoratus in the Upper Paraná River basin,Brazil

The aim of this study is to report the presence of a three non‐native hybrid long‐whiskered catfishes (family Pimelodidae) in the Upper Paraná River basin, Brazil. Genetic analyses demonstrated that the three presumptive hybrids were a result of the crossbreeding of Pseudoplatystoma reticulatum (central Amazonas River basin and Lower Paraná River) and Leiarius marmoratus (Amazonas, Essequibo and Orinoco rivers), producing a hybrid commonly known in Brazil as cachandiá. The potential threat to biodiversity, due to possible genetic contamination, competition and predation of wild stocks, of such artificially produced hybrid fishes is discussed.     

Photosynthesis and water‐use efficiency: A comparison between invasive (exotic) and non‐invasive (native) species

Abstract Invasive species have been hypothesized to out‐compete natives though either a Jack‐of‐all‐trades strategy, where they are able to utilize resources effectively in unfavourable environments, a master‐of‐some, where resource utilization is greater than its competitors in favourable environments, or a combination of the two (Jack‐and‐master). We examined the invasive strategy of Berberis darwinii in New Zealand compared with four co‐occurring native species by examining germination, seedling survival, photosynthetic characteristics and water‐use efficiency of adult plants, in sun and shade environments. Berberis darwinii seeds germinated more in shady sites than the other natives, but survival was low. In contrast, while germination of B. darwinii was the same as the native species in sunny sites, seedling survival after 18 months was nearly twice that of the all native species. The maximum photosynthetic rate of B. darwinii was nearly double that of all native species in the sun, but was similar among all species in the shade. Other photosynthetic traits (quantum yield and stomatal conductance) did not generally differ between B. darwinii and the native species, regardless of light environment. Berberis darwinii had more positive values of δ¹³C than the four native species, suggesting that it gains more carbon per unit water transpired than the competing native species. These results suggest that the invasion success of B. darwinii may be partially explained by combination of a Jack‐of‐all‐trades scenario of widespread germination with a master‐of‐some scenario through its ability to photosynthesize at higher rates in the sun and, hence, gain a rapid height and biomass advantage over native species in favourable environments.     

Roles of non‐native hydrogen‐bonding interaction in helix‐coil transition of a single polypeptide as revealed by comparison between Gō‐like and non‐Gō models

To explore the role of non‐native interactions in the helix‐coil transition, a detailed comparison between a Gō‐like model and a non‐Gō model has been performed via lattice Monte Carlo simulations. Only native hydrogen bonding interactions occur in the Gō‐like model, and the non‐native ones with sequence interval more than 4 is also included into the non‐Gō model. Some significant differences between the results from those two models have been found. The non‐native hydrogen bonds were found most populated at temperature around the helix‐coil transition. The rearrangement of non‐native hydrogen bonds into native ones in the formation of α‐helix leads to the increase of susceptibility of chain conformation, and even two peaks of susceptibility of radius of gyration versus temperature exist in the case of non‐Gō model for a non‐short peptide, while just a single peak exists in the case of Gō model for a single polypeptide chain with various chain lengths. The non‐native hydrogen bonds have complicated the temperature‐dependence of Zimm‐Bragg nucleation constant. The increase of relative probability of non‐native hydrogen bonding for long polypeptide chains leads to non‐monotonous chain length effect on the transition temperature. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Not all non‐natives are equally unequal: reductions in herbivore β‐diversity depend on phylogenetic similarity to native plant community

Effects of host plant α‐ and β‐diversity often confound studies of herbivore β‐diversity, hindering our ability to predict the full impact of non‐native plants on herbivores. Here, while controlling host plant diversity, we examined variation in herbivore communities between native and non‐native plants, focusing on how plant relatedness and spatial scale alter the result. We found lower absolute magnitudes of β‐diversity among tree species and among sites on non‐natives in all comparisons. However, lower relative β‐diversity only occurred for immature herbivores on phylogenetically distinct non‐natives vs. natives. Locally in that comparison, non‐native gardens had lower host specificity; while among sites, the herbivores supported were a redundant subset of species on natives. Therefore, when phylogenetically distinct non‐natives replace native plants, the community of immature herbivores is likely to be homogenised across landscapes. Differences in communities on closely related non‐natives were subtler, but displayed community shifts and increased generalisation on non‐natives within certain feeding guilds.     

Restored native prairie supports abundant and species‐rich native bee communities on conventional farms

Native pollinators are increasingly needed on conventional farms yet rarely fostered via management. One solution is habitat restoration in marginal areas, but colonization may be constrained if resident pollinator richness is low or if restored areas fail to provide sufficient floral or nesting resources. We quantified restoration outcomes for native bees, and associated resources, on three conventional farms with forb‐grass prairie plantings on marginal areas of varying sizes, in a heavily farmed region of central North America. We tested bee abundance and richness in restored prairie versus the dominant habitats of the region—crops, forest remnants, and edges of fields and roads. Restored prairie supported 2× more species (95 of 119 total species) and 3× more bees (72% of captured individuals) compared to the other cover types. All richness and abundance differences among habitat types were associated with higher floral resources in restored prairie. Thirty percent of the bee species were unique to prairie, consistent with long‐distance dispersal but begging the question of origin given the absence of prairie regionally. Our results suggest that road and field edges may be the source, as these areas had more floral and nesting resources than forest or crop fields combined and supported 55% of all species despite covering only approximately 5% of the sampled farms. Habitat scarcity is not the only constraint on native bees in agricultural landscapes, with increasing concern over disease and chemicals. However, we observed that restored areas on marginal lands of conventional farms can support abundant and species‐rich populations of native bees.     

Identifying potentially invasive non‐native marine and brackish water species for the Arabian Gulf and Sea of Oman

Invasive non‐native species (NNS) are internationally recognized as posing a serious threat to global biodiversity, economies and human health. The identification of invasive NNS is already established, those that may arrive in the future, their vectors and pathways of introduction and spread, and hotspots of invasion are important for a targeted approach to managing introductions and impacts at local, regional and global scales. The aim of this study was to identify which marine and brackish NNS are already present in marine systems of the northeastern Arabia area (Arabian Gulf and Sea of Oman) and of these which ones are potentially invasive, and which species have a high likelihood of being introduced in the future and negatively affect biodiversity. Overall, 136 NNS were identified, of which 56 are already present in the region and a further 80 were identified as likely to arrive in the future, including fish, tunicates, invertebrates, plants and protists. The Aquatic Species Invasiveness Screening Kit (AS‐ISK) was used to identify the risk of NNS being (or becoming) invasive within the region. Based on the AS‐ISK basic risk assessment (BRA) thresholds, 36 extant and 37 horizon species (53.7% of all species) were identified as high risk. When the impact of climate change on the overall assessment was considered, the combined risk score (BRA+CCA) increased for 38.2% of all species, suggesting higher risk under warmer conditions, including the highest‐risk horizon NNS the green crab Carcinus maenas, and the extant macro‐alga Hypnea musciformis. This is the first horizon‐scanning exercise for NNS in the region, thus providing a vital baseline for future management. The outcome of this study is the prioritization of NNS to inform decision‐making for the targeted monitoring and management in the region to prevent new bio‐invasions and to control existing species, including their potential for spread.     

LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

Unexpected side effects in biocrust after treating non‐native plants using carbon addition

Carbon addition has been proposed as an alternative to herbicide and manual removal methods to treat non‐native plants and reduce non‐target effects of treatments (e.g. impacts on native plants; surface disturbance). On Mojave Desert pavement and biocrust substrates after experimental soil disturbance and carbon addition (1,263 g C/m² as sucrose), we observed declines in lichens and moss cover in sucrose‐treated plots. To further explore this unforeseen potential side effect of using carbon addition as a non‐native plant treatment, we conducted biocrust surveys 5 and 7 years after treatments, sampled surface soils to observe if treatments additionally affected soil filamentous cyanobacteria, and conducted laboratory trials testing the effects of different levels of sucrose on cyanobacteria and desert mosses. Sucrose addition to biocrust plots reduced lichen and moss cover by 33–78% and species richness by 40–80%. Sucrose reduced biocrust cover in biocrust plots to levels similarly detected in pavement plots (<1%). While cyanobacteria in the field did not appear to be affected by sucrose, laboratory tests showed negative effects of sucrose on both cyanobacteria and mosses. Cyanobacteria declined by 41% 1 month after exposure to 5.4 g C/m² equivalent solutions. We detected injury to photosynthesis in mosses after 96 hour exposure to 79–316 g C/m² equivalent solutions. Caution is warranted when using carbon addition, at least in the form and concentration of sucrose, as a treatment for reducing non‐native plants on sites where conserving biocrust is a goal.