Molecular phylogeny of Sterrhinae moths (Lepidoptera: Geometridae): towards a global classification

A multigene phylogenetic study was carried out to test current, mostly morphology-based hypotheses on Sterrhinae phylogeny with additional material included from further geographical areas and morphologically different lineages. A maximum likelihood analysis (11 molecular markers and 7665 bp) was conducted on 76 species and 41 genera using iq-tree software. The resulting phylogenetic hypothesis is well resolved and branches have high support values. Results generally agree with earlier hypotheses at tribal levels and support the hypothesis that Sterrhinae comprises two major lineages. Based on the molecular phylogeny and extensive morphological examination, nine tribes are considered valid and the following taxonomic changes are introduced to recognize monophyletic groups: Mecoceratini Guenée, 1858 (= Ametridini Prout, 1910) is transferred from Desmobathrinae to Sterrhinae, and it is considered valid at tribal level new classification ; Haemaleini Sihvonen & Brehm is described as a new tribe and deemed sister to Scopulini + Lissoblemmini; Lissoblemmini Sihvonen & Staude is described as a new tribe and sister to Scopulini; Lythriini Herbulot, 1962 is now a junior synonym of Rhodometrini Agenjo, 1952 syn.n. ; and Rhodostrophiini Prout, 1935 is now a junior synonym of Cyllopodini Kirby, 1892 syn.n. In addition, 48 taxa are transferred from other geometrid subfamilies to Sterrhinae, or within Sterrhinae from one tribe to another, or they are classified into a tribe for the first time, or a new genus classification is proposed. The results demonstrate the limited explanatory power of earlier classifications, particularly at the tribal level. This is probably a result of earlier classifications being based on superficial characters and biased towards the European and North American fauna. The species richness and distribution of Sterrhinae and its constituent tribes are reviewed, showing that the globally distributed Sterrhinae are most diverse in the Neotropics (31% of global fauna). They are species-rich in the Palaearctic (22%), Afrotropics (19%) and Indo-Malay (16%) regions, whereas they are almost absent in Oceania (1%). In terms of the described fauna, the most species-rich tribes are Scopulini (928 species), Sterrhini (876 species) and Cosymbiini (553 species), all of which have a cosmopolitan distribution. Mecoceratiini and Haemaleini are almost entirely Neotropical. Timandrini and Lissoblemmini, by contrast, are absent in the Neotropics. We present a revised classification of the global Sterrhinae fauna, which includes about 3000 putatively valid species, classified into nine tribes and 97 genera. Four genera are of uncertain position within Sterrhinae. Our results highlight the compelling need to include more genera from a global perspective in molecular phylogenetic studies, in order to create a stable global classification for this subfamily. This published work has been registered on ZooBank, http://zoobank.org/urn:lsid:zoobank.org :pub:A66F5DDD-06D6-4908-893E-E8B124BB99B1.     

Local environment and connectivity are the main drivers of diatom species composition and trait variation in a set of tropical reservoirs

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Grazing tolerance of Gentianella amarella and other monocarpic herbs: why is tolerance highest at low damage levels?

Plants have adapted to compensate for the loss of vegetative biomass and reproductive potential caused by grazing. Shoot damage breaks down the correlative inhibition maintained by apical dominance. The consequent increased branching may lead to increased production of flowers and fruits in damaged plants, provided that enough resources, both in terms of meristems and nutrients, are available. In Gentianella amarella, the removal of the apex of the main stem (10% clipping) had no pronounced effect on branching and plant performance. In one of the two study populations, however, apically damaged plants produced more fruits than undamaged control plants. The plants also fully compensated for 50% removal of the main stem in terms of above-ground biomass, but their fruit production was reduced compared to control and apically damaged plants. After 75% clipping, fruit production was not significantly reduced compared to 50% clipping. Consequently, G. amarella showed highest tolerance in the presence of minor shoot damage. The pattern is qualitatively similar in some other monocarpic species (Gentianella campestris, Erysimum strictum and Rhinanthus minor). Multiple constraints as well as selective forces may shape these compensatory responses: (1) A lack of basal meristems may constrain tolerance of high damage levels. (2) Species with basal meristems may have a potential to tolerate major damage, but a shortage of resources or otherwise unfavourable growth conditions may constrain their compensatory ability. (3) It may be adaptive to have maximum tolerance of low and moderate damage levels if chemical defences reduce the risk of extensive shoot damage as well as the risk of repeated grazing. (4) The compensatory ability of monocarpic species may be affected by selective forces that favour fast vertical growth early in the season and unbranched architecture in undamaged conditions. Therefore, it is not the mere grazing history, but also other factors associated with growth conditions that are required to explain the variation in grazing tolerance.     

Use of sheep grazing in the restoration of semi‐natural meadows in northern Finland

Abstract. The biodiversity of species‐rich semi‐natural meadows is declining across Europe due to ceased management. In this study we aimed to find out how successfully the local species richness of an overgrown semi‐natural mesic meadow could be restored by sheep grazing after a long period of abandonment. The cover of vascular plant species in grazed plots and ungrazed exclosures was studied for five years and the responses of different functional plant groups were followed (herbs vs grasses, tall vs short species, species differing in flowering time, species representing different Grime's CSR strategies and species indicative of rich vs poor soil). Grazing increased species number by nearly 30%. On grazed plots the litter cover practically disappeared, favouring small herbs such as Rhinanthus minor, Ranunculus acris, Trifolium pratense and the grass Agrostis capillaris. Grazing decreased the cover of the late flowering tall herb Epilobium angustifolium but had no effect on the abundance of the early flowering tall herbs Anthriscus sylvestris or Geranium sylvaticum. We suggest that to succeed in restoration it is useful to determine the responses of different functional plant groups to grazing. Grassland managers need this information to optimize the methods and timing of management used in restoration. Additional management practices, such as mowing, may be needed in mesic meadows to decrease the dominance of tall species. The availability of propagules seemed to restrict further increase of species richness in our study area.     

Diatoms: unicellular surrogates for macroalgal community structure in streams?

As wholesale biodiversity assessment is often impractical, the use of surrogates that reflect the assemblage structure and diversity of other taxa has attracted increased attention. We sampled 47 boreal streams for diatoms and macroalgae and examined their assemblage patterns along major environmental gradients. Our main intention was to examine whether diatoms might be useful surrogates for macroalgae in boreal streams. We also assessed whether taxon richness and community composition provided similar insights into the patterns of cross-taxon concordance. According to canonical correspondence analysis, diatom distribution was most strongly related to water pH, conductivity, latitude and longitude, and macroalgal distribution to water pH and iron content, latitude and bed instability. In Mantel’s test, diatoms and macroalgae showed significant cross-taxon concordance. However, there was no significant correlation between taxon richness of the two algal groups, likely reflecting their differing responses to environmental variables. We found evidence that although diatoms and macroalgae are partly controlled by different environmental factors, they are segregated rather similarly along latitude and a few environmental gradients such as water pH and iron content. We conclude that, at least at broad geographical extents and in small streams, diatoms reflect the structure of the macroalgal community and are therefore useful surrogates for cost-effective biomonitoring of algal communities in streams.     

Taxonomic and functional diversity covary in rock pool microalgal communities despite their different drivers

Local biodiversity has traditionally been estimated with taxonomic diversity metrics such as species richness. Recently, the concept of biodiversity has been extended beyond species identity by ecological traits determining the functional role of a species in a community. This interspecific functional diversity typically responds more strongly to local environmental variation compared with taxonomic diversity, while taxonomic diversity may mirror more strongly dispersal processes compared with functional metrics. Several trait‐based indices have been developed to measure functional diversity for various organisms and habitat types, but studies of their applicability on aquatic microbial communities have been underrepresented. We examined the drivers and covariance of taxonomic and functional diversity among diatom rock pool communities on the Baltic Sea coast. We quantified three taxonomic (species richness, Shannon''s diversity, and Pielou''s evenness) and three functional (functional richness, evenness, and divergence) diversity indices and determined abiotic factors best explaining variation in these indices by generalized linear mixed models. The six diversity indices were highly collinear except functional evenness, which merely correlated significantly with taxonomic evenness. All diversity indices were always explained by water conductivity and temperature–sampling month interaction. Taxonomic diversity was further consistently explained by pool distance to the sea, and functional richness and divergence by pool location. The explained variance in regression models did not markedly differ between taxonomic and functional metrics. Our findings do not clearly support the superiority of neither set of diversity indices in explaining coastal microbial diversity, but rather highlight the general overlap among the indices. However, as individual metrics may be driven by different factors, the greatest advantage in assessing biodiversity is nevertheless probably achieved with a simultaneous application of the taxonomic and functional diversity metrics.     

Single-label time-resolved luminescence assay for estrogen receptor--ligand binding

Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR–FRET) assays provide high sensitivity due to low background signal. The TR–FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)–ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu–E₂) is quenched using soluble quencher molecules. The luminescence signal of Eu–E₂ on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu–E₂ from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8–9.2) were clearly higher than the S/B values for the FP assay (1.3–1.5). A K_d value of 30 nM was calculated for binding of Eu–E₂ to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.     

Incidence of asthma in children in Tuzla Canton--Bosnia and Herzegovina

Asthma is one of the most common chronic diseases whose incidence shows constant growth in childhood. The objective of this work was to look into asthma incidence in children in relation to their age group and sex in a retrospective study, at Tuzla Canton area. The study comprised children of both sexes, age 0-14 who fell sick with asthma within the period from January 1st 2003 to December 31st 2007. The overall incidence and the incidence in relation to age group and sex was calculated as the number of children suffering from asthma, within the age group 0-14 years per 1000 children of the same age group in the Tuzla Canton. Asthma was diagnosed in 277 children (66.1% male and 33.9% female). The difference between asthma frequency in boys and girls was significant (chi2 = 56.16; df = 1; p < 0.0001). The average difference in proportion between the boys and girls was 32.2% (95% CI = 24.32-40.08). From this sample group the boys had a 3.8 times greater risk (OR = 3.79; %95 CI = 2.67-5.39) of contracting asthma. The average rate of incidence of asthma for both sexes in the observed period was 0.67/1000 (95% CI; 0.6-0.7; for boys 0.86/1000; for girls 0.47/1000). There was a statistically significantly higher incidence of asthma in boys in relation to girls (t = 6.3836, df = 32; p < 0.0001). The epidemiological data obtained could be useful for early detection and adequate treatment of children with asthma in the mentioned area.     

μABC: a systematic microsecond molecular dynamics study of tetranucleotide sequence effects in B-DNA

We present the results of microsecond molecular dynamics simulations carried out by the ABC group of laboratories on a set of B-DNA oligomers containing the 136 distinct tetranucleotide base sequences. We demonstrate that the resulting trajectories have extensively sampled the conformational space accessible to B-DNA at room temperature. We confirm that base sequence effects depend strongly not only on the specific base pair step, but also on the specific base pairs that flank each step. Beyond sequence effects on average helical parameters and conformational fluctuations, we also identify tetranucleotide sequences that oscillate between several distinct conformational substates. By analyzing the conformation of the phosphodiester backbones, it is possible to understand for which sequences these substates will arise, and what impact they will have on specific helical parameters.     

Quantitative Chemical Proteomics Identifies Novel Targets of the Anti-cancer Multi-kinase Inhibitor E-3810

Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts).E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (K_{d imm}) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and—in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer—cellular assays.Taken together, the success of our strategy—which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry—in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.The “target deconvolution” process, namely, the identification and characterization of proteins bound by a drug of interest (1), is a crucial step in drug development that allows definition of the compound selectivity and the early detection of potential side effects. Target deconvolution can be achieved by means of systematic in vitro biochemical assays measuring the ability of the drug to interact with candidate binders and, if they are enzymes, interfere with their activity. An alternative approach is chemical proteomics (chemoproteomics), which combines affinity chromatography and proteomic techniques (2, 3). Up-to-date chemical proteomics essentially consists of three main steps: (i) drug immobilization on a solid phase; (ii) drug affinity chromatography to capture drug targets in complex protein mixtures, such as cell or tissue lysates; and (iii) mass spectrometry (MS)-based¹ identification of the proteins retained by the immobilized drug (4–6).In chemical proteomics, the affinity chromatography step is typically performed under mild conditions, to allow the identification of all possible natural binders. The drawback of using mild, non-denaturing conditions is the significant number of proteins nonspecifically binding to the solid phase, which, once identified via MS, can be difficult to discern from genuine drug targets. The relatively high number of such nonspecific binders has limited the widespread use of this strategy.More recently, the development and implementation of quantitative strategies in proteomics based on the use of differentially stable isotopes to label proteomes from distinct functional states, together with significant technological and instrumental developments in the MS field concerning sensitivity and throughput, have largely allowed this limitation to be overcome. One of the most popular labeling techniques is stable isotope labeling by amino acids in cell culture (SILAC) (7). In SILAC, dividing cells are cultured in media supplemented with amino acids containing stable isotopic variants of carbon (¹²C/¹³C), nitrogen (¹⁴N/¹⁵N), or hydrogen (¹H/²H), which are incorporated into newly synthesized proteins during cell division. When extensive labeling (>98%) of cells is achieved upon the appropriate number of replications, light and heavy cells are differentially treated (e.g. exposed to drug versus vehicle), mixed in equal proportion, and subjected to proteomics analysis by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Peptides from the two functional states can be distinguished by their specific delta mass values, and their intensity ratio in MS spectra is directly proportional to the relative abundance of the corresponding proteins in the initial protein extract. Robust analysis of SILAC data is possible with dedicated software, such as MaxQuant (8). The application of SILAC strategies to interactomic studies is an efficient means of discerning specific from background binders (9). When applied to chemical proteomics, quantitative proteomics is crucial, as it offers quality filters to discern genuine drug interactors from proteins binding to the solid phase, with the use of different experimental setups (4, 5).In this study, we successfully coupled SILAC with chemical proteomics to carry out an unbiased screening of protein interactors of the anti-cancer drug E-3810, currently in Phase II clinical trials. E-3810 is a novel multi-kinase inhibitor, a class of targeted drug that comprises different molecules currently used in clinical practice (e.g. imatinib, dasatinib, sunitinib, sorafenib) (10). E-3810 exhibits both anti-tumor and anti-angiogenic properties (11). In preclinical studies, E-3810 showed broad anti-tumor activity in vivo, when used as monotherapy in a variety of human xenografts, or in conjunction with conventional chemotherapy (11, 12).Cellular vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) are the principal targets of E-3810, as previously demonstrated by in vitro kinase assays, which showed that E-3810 inhibited VEGFR-1, -2, and -3 and FGFR-1 and -2 in the nanomolar range (11). Studies performed on several kinase inhibitors demonstrated that these molecules can elicit pleiotropic effects not easily explained by the sole inhibition of their known targets (13, 14). These effects are in most cases due to an inhibitory activity of the drug on additional kinase targets not tested in vitro that may lead to synergistic anti-cancer effects or undesirable toxicity. This could also be the case for E-3810, which was shown to inhibit in vitro additional kinase targets with high affinity, and which is able to inhibit the growth of tumor cells expressing low to undetectable levels of VEGFRs/FGFRs, suggesting that its spectrum of target inhibition has not been fully explored (11).We thus established a SILAC-based chemical proteomic platform composed of a set of affinity chromatography experiments using E-3810 immobilized on agarose resin and incubated with SILAC-labeled extract from the ovarian cancer cell line A2780. We identified proteins interacting with the resin via MS and took advantage of SILAC-based protein quantitation to discern genuine from background binders and derive quantitative information about the specific interactions. Our findings demonstrate that additional targets of E-3810 exist and that these targets may contribute to the anticancer effect of E-3810.