Adapt or die—Response of large herbivores to environmental changes in Europe during the Holocene

Climate warming and human landscape transformation during the Holocene resulted in environmental changes for wild animals. The last remnants of the European Pleistocene megafauna that survived into the Holocene were particularly vulnerable to changes in habitat. To track the response of habitat use and foraging of large herbivores to natural and anthropogenic changes in environmental conditions during the Holocene, we investigated carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope composition in bone collagen of moose (Alces alces), European bison (Bison bonasus) and aurochs (Bos primigenius) in Central and Eastern Europe. We found strong variations in isotope compositions in the studied species throughout the Holocene and diverse responses to changing environmental conditions. All three species showed significant changes in their δ¹³C values reflecting a shift of foraging habitats from more open in the Early and pre‐Neolithic Holocene to more forest during the Neolithic and Late Holocene. This shift was strongest in European bison, suggesting higher plasticity, more limited in moose, and the least in aurochs. Significant increases of δ¹⁵N values in European bison and moose are evidence of a diet change towards more grazing, but may also reflect increased nitrogen in soils following deglaciation and global temperature increases. Among the factors explaining the observed isotope variations were time (age of samples), longitude and elevation in European bison, and time, longitude and forest cover in aurochs. None of the analysed factors explained isotope variations in moose. Our results demonstrate the strong influence of natural (forest expansion) and anthropogenic (deforestation and human pressure) changes on the foraging ecology of large herbivores, with forests playing a major role as a refugial habitat since the Neolithic, particularly for European bison and aurochs. We propose that high flexibility in foraging strategy was the key for survival of large herbivores in the changing environmental conditions of the Holocene.     

Demographic history has influenced nucleotide diversity in European Pinus sylvestris populations

To infer the role of natural selection in shaping standing genetic diversity, it is necessary to assess the genomewide impact of demographic history on nucleotide diversity. In this study we analyzed sequence diversity of 16 nuclear loci in eight Pinus sylvestris populations. Populations were divided into four geographical groups on the basis of their current location and the geographical history of the region: northern Europe, central Europe, Spain, and Turkey. There were no among-group differences in the level of silent nucleotide diversity, which was approximately 0.005/bp in all groups. There was some evidence that linkage disequilibrium extended further in northern Europe than in central Europe: the estimates of the population recombination rate parameter, rho, were 0.0064 and 0.0294, respectively. The summary statistics of nucleotide diversity in central and northern European populations were compatible with an ancient bottleneck rather than the standard neutral model.     

Air pollution distribution patterns in the San Bernardino Mountains of southern California: a 40-year perspective

Since the mid-1950s, native pines in the San Bernardino Mountains (SBM) in southern California have shown symptoms of decline. Initial studies in 1963 showed that ozone (O3) generated in the upwind Los Angeles Basin was responsible for the injury and decline of sensitive trees. Ambient O3 decreased significantly by the mid-1990s, resulting in decreased O3 injury and improved tree growth. Increased growth of trees may also be attributed to elevated atmospheric nitrogen (N) deposition. Since most of the N deposition to mixed conifer forest stands in the SBM results from dry deposition of nitric acid vapor (HNO3) and ammonia (NH3), characterization of spatial and temporal distribution of these two pollutants has become essential. Although maximum daytime O3 concentrations over last 40 years have significantly decreased (approximately 3-fold), seasonal means have been reduced much less (approximately 1.5-fold), with 2-week long means occasionally exceeding 100 ppb in the western part of the range. In the same area, significantly elevated concentrations of HNO3 and NH3, up to 17.5 and 18.5 microg/m3 as 2-week averages, respectively, have been determined. Elevated levels of O3 and increased N deposition together with long-term drought predispose the SBM forests to massive bark beetle attacks making them susceptible to catastrophic fires.     

Untangling the Echiniscus Gordian knot: paraphyly of the “arctomys group” (Heterotardigrada: Echiniscidae)

The genus Echiniscus C.A.S. Schultze, 1840, one of the earliest established and speciose tardigrade genera, has been hypothesized to be polyphyletic. Moreover, the presence or absence of trunk appendages, the type of cuticular sculpturing and body colour have been argued to hold taxonomic significance at the genus level in Echiniscus-line taxa. Here, by combining morphological and genetic analyses, we demonstrate that the so-called “arctomys group”, i.e. Echiniscus spp. lacking trunk appendages, comprises numerous separate evolutionary lineages within the family Echiniscidae. As a result, we erect five new echiniscid genera: Barbaria gen. nov. , presumably of Neotropical (Gondwanan) origin, previously classified as the Echiniscus bigranulatus group; the pantropical and subtropical Kristenseniscus gen. nov. (the tessellatus group), characterized by a peculiar subdivision of dorsal plates; Claxtonia gen. nov. (the wendti group), with large and evident endocuticular pillars in the form of polygons; Nebularmis gen. nov. (the reticulatus group), with an elusive dorsal sculpturing; and Viridiscus gen. nov. (the viridis group), with body colour ranging from light green through brownish to even almost black. Additionally, we briefly address appendaged Echiniscus s.s. and divide the genus into several groups based on dorsal plate sculpturing and suggest that these could also represent separate supraspecific entities.     

Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane "rafts" biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 micro, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.     

Kinin B1 receptor antagonists with multi-enzymatic resistance properties

The kinin B, receptor has been implicated in a variety of pathological states; therefore, potent, selective, and specific antagonists with prolonged duration of action in vivo are needed. Using R-715 (AcLys[D-beta-Nal(7),Ile(8)] desArg9BK) as a template, new peptides containing alpha-MePhe in position 5, Oic in position 2, and AcOrn instead of AcLys at the N-terminal were prepared and tested for their antagonist potency, their selectivity, and their specificity for the kinin B1 receptor. In vitro metabolic stabilities toward aminopeptidase M (from human plasma), aminopeptidase P (from human platelets), and angiotensin-converting enzyme (purified from rabbit lung) were also investigated. The results of this study indicate that the three modifications applied separately are as well tolerated as they are when present conjointly in the template R-715. Indeed, pA2 values of R-715 (ranging from 8.40 to 8.5) do not differ significantly from the analogues R-954 and R-955 (both ranging from 8.4 to 8.6) when measured at kinin B1 receptors from rabbit aortas and human umbilical veins. Moreover, the chemical modifications utilized in the peptides R-954 and R-955 have provided resistance against aminopeptidases M and P, as well as the angiotensin-converting enzyme, unlike the early (e.g., Lys[Leu8]desArg9BK) and more recent (e.g., R-715, B-9858) generations of B, receptor antagonists. Ongoing in vivo assays will validate the assumption that the analogues R-954 and R-955 have a prolonged duration of action.     

Glutathione S transferase M1 andT1 genotypes and susceptibility to smoking related larynx cancer

Susceptibility to smoking related larynx cancer has been suggested to be associated with genetically determined differences in the ability to detoxify carcinogens present in tobacco smoke. The genetic polymorphisms of glutathione S-transferases, involved in the metabolic inactivation of, for example, tobacco derived carcinogens, have been recognized as potential risk modifiers in various environmentally induced malignancies, including larynx cancer. We employed PCR-based methods to determine the distribution of the GSTM 1 and G STT1 null genotypes in 171 larynx cancer patients and 180 controls to examine further their potential role in individual susceptibility to this neoplasm. The GSTM 1 null genotype was found in 49 1 % of the cases and 57 7 % of the controls and the GSTT1 null genotype in 17 5 % of the cases and 21 7 % of the controls, respectively. Larynx cancer risk associated with the lack of GST M 1 (OR = 0 7; 95 % CI: 0 5-1 1) or GSTT1 (OR = 0 8; 95 % CI: 0 5-1 3) was not significantly affected by age, smoking status, or cancer progression. Although this study thus suggests no role for the G STM 1 and GSTT1 gene polymorphisms in individual susceptibility to smoking-related larynx cancer, due to its relatively small sample size more data are required before any definite conclusions can be drawn.     

Cytoskeletal Components Define Protein Location to Membrane Microdomains

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.Living cells need borders and molecular compartments for biochemical reactions and storage of metabolites. The plasma membrane therefore is a prerequisite for the evolution of different life forms. It consists of a phospholipid bilayer into which proteins and special lipid species such as sterols, sphingolipids, and glycolipids are inserted. The first complex model of plasma membrane was proposed in 1972 by Jonathan Singer and Garth Nicolson (1), replacing the concept of the plasma membrane as a strict protein–lipid–protein sandwich that was generally accepted until then. In Singer and Nicolson''s model, the cell membrane is a two-dimensionally oriented viscous solution in which the membrane constituents are orientated in the most thermodynamically favorable manner, hiding hydrophobic hydrocarbon chains inside the lipid bilayer and exposing polar and ionic groups to the aqueous phase. This fluid mosaic model also implied that membrane proteins as well as lipid components are distributed in a homogeneous lipid bilayer at long range, but they can form specific aggregates and phases at short range, which were also termed “lipid rafts” or membrane microdomains.Over the past 30 years, it has become evident that the plasma membrane is not such a homogeneous structure as it was initially proposed. We now know that the lipid bilayer is asymmetric (2) and that the free diffusion of membrane proteins is restricted by their interactions with intracellular and extracellular components (3). More recently, Simons and Ikonen suggested that large ordered phases, enriched with cholesterol and sphingolipids, emerge within the plasma membrane and that they function as platforms for enrichment of certain proteins while excluding others (4). This current membrane model suggests that the mixture of sterols and polar lipids within the plasma membrane can appear in two distinct phases: liquid disordered (L_d) and liquid ordered (L_o) phase (5). In this view, the so-called membrane microdomains are considered to be part of the L_o phase. Based on work on model membranes, it is suggested that lateral segregation of components into L_d and L_o phases occurs spontaneously (6) with the self-associating properties between sterols and highly saturated hydrocarbon chains of phopsho- and sphingolipids as the main driving force (7). Additionally, it is suggested that also specific lipid-protein and protein-protein interactions are essential for the formations of membrane domains as well as for stabilization of smaller nanodomains which subsequently may cause formation of larger platforms. In contrast to the animal cells, in plants these membrane microdomains seem to be rather immobile (8), possibly due to their attachment to the outer cell wall. More recently, it became obvious that membrane microdomains within a single cell are highly diverse and of different compositions (9). Generally, in the plant model, organisms'' plasma membrane microdomains turned out to be important in plant defense (10, 11), cell polarity (12, 13), and general signaling properties of the plasma membrane (14, 15).The cytoskeleton was identified as an essential cellular component with important roles in membrane topography, bordering, trafficking, and organelle movement (16). Single particle tracking in mammalian cells revealed that the transferrin receptor and macroglobulin receptor demonstrate normal Brownian diffusion but only within a specific membrane compartment (17). Two hypothetical models were proposed in order to explain this phenomenon (supplemental Fig. 1). Direct interactions between transmembrane proteins and cytoskeleton are suggested to creates a barrier, called “fence,” where cytosolic parts of transmembrane proteins collides with cytoskeletal components, limiting their diffusion to certain areas. These molecules can jump over the “fence” to a neighboring compartment, possibly due to the dynamic nature of the interaction of membrane proteins and cytoskeleton, where they are again temporally trapped (17). This phenomenon was recently described also in A. thaliana where the interplay between membrane microdomains and microtubules plays a role in secondary cell wall formation (reviewed in (18)). The second model assumes, additionally, that particular transmembrane proteins are anchored to and lined up along cytoskeleton and act as “pickets” to arrest free diffusion of other membrane components, including nontransmembrane proteins, within the enclosed compartment (19).For plants, the composition of these sterol-rich membranes phases was analyzed in several biochemical studies (14, 20–22). Thereby, low-density preparations of plasma membrane fractions after treatment with nonionic detergents (DRM¹ fractions) were considered as a biochemical representation enriched in cellular membrane ordered phases or microdomains. Proteomic studies in mammalian cells consistently reported that the DRM fraction is highly enriched with several cytoskeletal proteins such as actin, tubulin, myosin, dynamin, actinin, and supervillin (23–25). Additionally, the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a lipid connecting the plasma membrane to actin filaments, was also significantly elevated in DRM preparations (26). Treatment with microtubule and actin depolymerizing agent results in drastic loss of many signaling proteins from these DRM fractions prepared from adult rat cardiac myocytes (27) or human embryonic retinal cells (28).Based on this knowledge, we propose two hypothetical models for the relationship between cytoskeleton and membrane microdomains for plant cells: (i) Actin filaments and microtubules could be important in the membrane phase separation or formation of the membrane microdomains themselves. In this case, disruption of the cytoskeleton would cause a lack of phase segregation in the plasma membrane. (ii) The cytoskeleton is only important for the incorporation of specific protein into the sterol-enriched regions but not for the general formation of these phase separations. This view implies that phase separations or membrane microdomains would still be present after cytoskeleton disruption but their protein composition can be different. Another possible scenario is (iii) that cytoskeletal elements serve as anchors for membrane microdomains at particular position in the plasma membrane, so the absence of these anchors would cause the increased mobility of microdomains (supplemental Fig. 1).The primary aim of this study was to characterize the interplay between cytoskeletal components and different membrane phases (microdomains) in A. thaliana suspension cell cultures. To reach this goal, biochemical and proteomic approaches were combined with confocal microscopy and activity assays measuring the influence of actin or tubulin disruption on the composition, localization, and biochemical properties of the sterol-enriched membrane microdomains. Thereby, for biochemical analyses, low-density detergent-resistant membrane fractions are analyzed as containing cellular sterol-rich membrane compartments.     

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression.