Proposal for a simple hydromorphological habitat survey method for freshwater bivalve (Unionidae) inventories

Numerous species of Unionidae are presently threatened by anthropogenic impacts on freshwater ecosystems. Field inventories, based on reliable presence/absence observations, are urgently needed to improve their protection. Such observations should be comparable between sites and collected with minimal surveying effort. Here, we demonstrate a strategy that minimises sampling effort while maximising inventory efficiency, formulated using hydromorphological data from 26 river sites sampled in north-eastern France between 2009 and 2010. Our findings indicate that a comprehensive visual survey of seven x river width with a bathyscope confirmed unionid presence in 96 % of cases, and presence of all unionid species in 88 % of cases. A further seven x width search increased this latter figure to 96 %, while a third (=21 × width) ensured that all species were registered in all rivers surveyed. Based on these results, we recommend that surveyors first undertake an initial seven x river width visual survey to confirm unionid presence. If no Unionidae are observed over this distance, sampling ceases and the site is marked negative. If at least one Unionidae is observed over this distance, an additional upstream stretch of the same length is surveyed with identical sampling effort. If at least one new species is observed within this second stretch, then a third and final stretch can be surveyed. This method is discussed in the light of representativeness of hydromorphological habitats (e.g. pool spacing and meander wavelength).     

Beneficial fitness effects are not exponential for two viruses

The distribution of fitness effects for beneficial mutations is of paramount importance in determining the outcome of adaptation. It is generally assumed that fitness effects of beneficial mutations follow an exponential distribution, for example, in theoretical treatments of quantitative genetics, clonal interference, experimental evolution, and the adaptation of DNA sequences. This assumption has been justified by the statistical theory of extreme values, because the fitnesses conferred by beneficial mutations should represent samples from the extreme right tail of the fitness distribution. Yet in extreme value theory, there are three different limiting forms for right tails of distributions, and the exponential describes only those of distributions in the Gumbel domain of attraction. Using beneficial mutations from two viruses, we show for the first time that the Gumbel domain can be rejected in favor of a distribution with a right-truncated tail, thus providing evidence for an upper bound on fitness effects. Our data also violate the common assumption that small-effect beneficial mutations greatly outnumber those of large effect, as they are consistent with a uniform distribution of beneficial effects.     

Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus

Tachylectin-2, isolated from large granules of the hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), is a 236 amino acid protein belonging to the lectins. It binds specifically to N-acetylglucosamine and N-acetylgalactosamine and is a part of the innate immunity host defense system of the horseshoe crab. The X-ray structure of tachylectin-2 was solved at 2.0 A resolution by the multiple isomorphous replacement method and this molecular model was employed to solve the X-ray structure of the complex with N-acetylglucosamine. Tachylectin-2 is the first protein displaying a five-bladed beta-propeller structure. Five four-stranded antiparallel beta-sheets of W-like topology are arranged around a central water-filled tunnel, with the water molecules arranged as a pentagonal dodecahedron. Tachylectin-2 exhibits five virtually identical binding sites, one in each beta-sheet. The binding sites are located between adjacent beta-sheets and are made by a large loop between the outermost strands of the beta-sheets and the connecting segment from the previous beta-sheet. The high number of five binding sites within the single polypeptide chain strongly suggests the recognition of carbohydrate surface structures of pathogens with a fairly high ligand density. Thus, tachylectin-2 employs strict specificity for certain N-acetyl sugars as well as the surface ligand density for self/non-self recognition.     

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C₁₃ apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.In plants, the synthesis of carotenoids is plastid localized, with the plastid type determining their function (Ruiz-Sola and Rodríguez-Concepción, 2012; Nisar et al., 2015). In nonphotosynthetic chromoplasts, carotenoids and their volatile derivatives attract pollinating insects or zoochoric animals. Here, carotenoids are sequestered in diverse suborganellar structures, which can be tubulous, globulous, membranous, or crystalline (Sitte et al., 1980; Egea et al., 2010). In chloroplasts, carotenoids are present in light-harvesting complex proteins and photosynthetic reaction centers. They extend the light spectrum used for photosynthetic energy transformation and act photoprotectively because of their ability to quench excitation energy from singlet- or triplet-state chlorophylls, thereby decreasing the risk that singlet oxygen forms (Niyogi, 1999; Demmig-Adams and Adams, 2002). Furthermore, the regulated epoxidation and deepoxidation of zeaxanthin in the xanthophyll cycle contribute to the nonphotochemical quenching of energy (Niyogi, 1999; Ballottari et al., 2014). In contrast to these processes, which maintain carotenoid integrity, carotenoids are also capable of chemically quenching singlet oxygen by their own oxidation, which is accompanied by the release of various carotenoid degradation products (Ramel et al., 2012a, 2013).The various functions of carotenoids require their dynamic qualitative and quantitative tuning in response to environmental conditions to attain and maintain adequate steady-state concentrations. These include both the regulation of their synthesis and the formation, release, or disposal of their breakdown products. The synthesis of carotenoids is initiated by the condensation of two molecules of geranylgeranyl diphosphate to form phytoene catalyzed by the enzyme phytoene synthase (PSY), which is considered as the rate-limiting enzyme (von Lintig et al., 1997; Li et al., 2008; Rodríguez-Villalón et al., 2009; Welsch et al., 2010; Zhou et al., 2015). In plants, two desaturases, phytoene desaturase and ζ-carotene desaturase, and two carotene cis-trans-isomerases convert the colorless phytoene into the red-colored all-trans-lycopene (Isaacson et al., 2002; Park et al., 2002; Chen et al., 2010; Yu et al., 2011). Two lycopene cyclases introduce either β- or ε-ionone rings, yielding α-(ε,β-)-carotene and β-(β,β)-carotene. In Arabidopsis (Arabidopsis thaliana), four enzymes hydroxylate carotenes with partially overlapping substrate specificity (Kim et al., 2009). Two nonheme iron-dependent β-carotene hydroxylases (BCH), BCH1 and BCH2, convert β-carotene into zeaxanthin. The second set of hydroxylases, cytochrome P450 (CYP)97A3 and CYP97C1, prefers α-carotene and produces zeinoxanthin and lutein, respectively. Absence of each cytochrome P450 hydroxylase constitutes a distinct phenotype, named lutein deficient5 (lut5) for CYP97A3 deficiency and lut1 for CYP97C1 deficiency, characterized by altered pigment compositions and the accumulation of monohydroxylated intermediates, whereas deficiency in BCH1 and BCH2 does not affect the pigment composition.In green tissues, photooxidative destruction seemingly predominates and consumes carotenoids (Simkin et al., 2003). Moreover, ¹⁴CO₂ pulse-chase experiments with Arabidopsis leaves identified α- and β-carotene as the main targets for photooxidation, whereas xanthophylls were less affected (Beisel et al., 2010). Oxidation assays with β-carotene showed epoxy- and peroxy-derivatives as the main primary products, which however, undergo additional reactions, yielding more stable degradation products that are, in part, the same apocarotenals/ones as those being produced enzymatically (Ramel et al., 2012a, 2013).In Arabidopsis, genes coding for carotenoid cleaving enzymes (carotenoid cleavage dioxygenases [CCDs]) form a small gene family comprising nine members, five of which are attributed to the synthesis of abscisic acid (ABA; nine-cis-epoxycarotenoid cleavage dioxygenases [NCEDs]; AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9; Iuchi et al., 2001; Tan et al., 2003), whereas two are committed to strigolactone biosynthesis (CCD7/MORE AXILLARY GROWTH3 [MAX3] and CCD8/MAX4; Alder et al., 2012; Bruno et al., 2014). Orthologs of CCD1 are involved in the generation of volatile apocarotenoids contributing to flower scents and aroma production (e.g. saffron [Crocus sativus; Rubio et al., 2008; Frusciante et al., 2014] and tomato [Solanum lycopersicum; Simkin et al., 2004]), whereas CCD4 enzymes are involved in citrus peel and chrysanthemum (Chrysanthemum morifolium) petal coloration (Ohmiya et al., 2006; Rodrigo et al., 2013). Recent analysis of Arabidopsis mutants revealed a major function of CCD4 in regulating seed carotenoid content with only a minor contribution of CCD1 (Gonzalez-Jorge et al., 2013). Moreover, CCD4 activity was required for the synthesis of an apocarotenoid-derived signaling molecule involved in leaf development and retrograde gene expression (Avendaño-Vázquez et al., 2014).Elevated carotenoid pathway flux caused by PSY overexpression increases carotenoid accumulation in various nongreen tissues, such as tomato fruits, canola (Brassica napus) seeds, cassava (Manihot esculenta) roots, and rice (Oryza sativa) endosperm (Shewmaker et al., 1999; Ye et al., 2000; Fraser et al., 2002; Welsch et al., 2010). Similarly, the constitutive overexpression of PSY in Arabidopsis results in dramatically increased carotenoid amounts accumulating as crystals in nongreen tissues, such as roots and callus, yielding β-carotene as the main product (Maass et al., 2009). However, leaves of the very same plants do not show altered pigment composition, and phytoene or other pathway intermediates are not detected. Similarly, increased levels of active PSY protein achieved though overexpression of the ORANGE protein exclusively affect carotenoid amounts in roots but not in leaves (Zhou et al., 2015). Leaves from constitutively PSY-overexpressing tomato and tobacco (Nicotiana tabacum) plants are also reported to show only slightly increased carotenoid levels compared with the wild-type control (Fray et al., 1995; Busch et al., 2002). These contrasting responses of leaves versus nongreen tissues to elevated pathway flux suggest fundamental differences in the modes of carotenoid formation and/or degradation.In this work, we identified xanthophyll-derived apocarotenoid glycosides (AGs) in Arabidopsis leaves that increase upon higher pathway flux. This suggests that apocarotenoid glycosylation functions as a valve regulating carotenoid steady-state levels in leaves. The analysis of Arabidopsis mutants enabled us to conclude on potential precursor carotenoids and assess the contribution of carotenoid cleavage enzymes on their formation. Moreover, apocarotenoids but not the identified glycosides were increased in carotenoid-hyperaccumulating roots, indicating tissue-specific different modes of carotenoid turnover regulation.     

Statistical tests for intra-tumour clonal co-occurrence and exclusivity

Tumour progression is an evolutionary process in which different clones evolve over time, leading to intra-tumour heterogeneity. Interactions between clones can affect tumour evolution and hence disease progression and treatment outcome. Intra-tumoural pairs of mutations that are overrepresented in a co-occurring or clonally exclusive fashion over a cohort of patient samples may be suggestive of a synergistic effect between the different clones carrying these mutations. We therefore developed a novel statistical testing framework, called GeneAccord, to identify such gene pairs that are altered in distinct subclones of the same tumour. We analysed our framework for calibration and power. By comparing its performance to baseline methods, we demonstrate that to control type I errors, it is essential to account for the evolutionary dependencies among clones. In applying GeneAccord to the single-cell sequencing of a cohort of 123 acute myeloid leukaemia patients, we find 1 clonally co-occurring and 8 clonally exclusive gene pairs. The clonally exclusive pairs mostly involve genes of the key signalling pathways.     

Stream community structure in relation to spatial variation: the influence of mesohabitat characteristics

Community structure of benthic macroinvertebrates was studied in six first- through fourth-order streams in northeast France, to elucidate changes in richness, abundance, diversity and evenness of mesohabitat assemblages as a function of environmental conditions. Patch samples were subjected to multivariate analyses to determine: (i) relationships among seven indices describing community structure (structure parameters); (ii) relationships among seven environmental variables; (iii) the relationship between community structure and environmental characteristics of patches. Faunal data showed that indices measuring the distribution of individuals among taxa (evenness, dominance) and richness are prominent in describing the structure of macroinvertebrate communities of mesohabitats. The analysis of environmental data demonstrated a major differentiating ability of current velocity and strong inter-relations among in-stream hydraulic-dependent parameters in structuring the mesohabitat environment. The co-structure (= relationship) between community organization and environmental variables indicated that substrate may be a primary determinant of community structure. Current velocity and water depth emerged as secondary factors. Trends in community structure were closely related to the spatial variability of mesohabitats. Species richness increased with habitat heterogeneity. Total abundance increased with trophic potentialities of patches. Equitability and diversity seemed to increase with patch stability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene.

Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M. Hummel, D. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417). These RNAs have the same 5' and 3' ends. The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame. The smaller RNA has one internal splice which maintains the same open reading frame. Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J. Virol. 49:413-417). The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties. Antisera raised in rabbits to the protein expressed in E. coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame. The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement. This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody. We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.