The effect of the dominant grass <Emphasis Type="Italic">Festuca rupicola</Emphasis> on the establishment of rare forbs in semi-dry grasslands

Establishment is the most sensitive process in the life-cycle of plant species, and each stage – from germination to survival and growth – can be affected by environmental factors and plant traits. We hypothesized that the dominant tussock grass Festuca rupicola negatively affects forb establishment in semi-dry grasslands where it has recently expanded into. Moreover, we expected that seedling recruitment is affected by grass density and seed size, with larger seeded forbs being more successful in areas of higher grass density. In a garden experiment, we sowed seven forb individuals of differing seed size (smallest to largest: Veronica spicata < Dianthus carthusianorum < Plantago media < Biscutella laevigata < Filipendula vulgaris < Scabiosa ochroleuca < Sanguisorba minor) into pots with zero, one, two and three grass individuals, and assessed germination, survival and growth over one year. As expected, increasing grass density negatively affected germination, survival and growth of forbs; however, contrary to our expectation, seed size did not influence any of parameters measured. The response of each individual species varied from zero or weak to strong with respect to grass density. Festuca rupicola therefore acts as a strong competitor against the establishment of forbs, irrespective of their seed size, and its spread lowers species diversity in semi-dry grasslands.     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.     

Xylene‐Bridged Phosphaviologen Oligomers and Polymers as High‐Performance Electrode‐Modifiers for Li‐Ion Batteries

Lithium‐ion batteries are one of the most common forms of energy storage devices used in society today. Due to the inherent limitations of conventional Li‐ion batteries, organic materials have surfaced as potentially suitable electrode alternatives with improved performance and sustainability. Viologens and phosphaviologens in particular, are strong electron‐accepting materials with excellent kinetic properties, making them suitable candidates for battery applications. In this paper, new polymeric species of the latter moieties are reported that lead to improved electrode stability and device performance. The performance of the phosphaviologen is further enhanced through the utilization of both redox steps, allowing for good performance proof‐of‐concept hybrid organic/Li‐ion batteries. This opens the potential for more sustainable and improved battery performance for use in current energy applications.     

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1–4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4–7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6, 8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more preserved biological materials that are more amenable to downstream analytical assays. One example of this is the controversial case of bone and soft-tissue preservation from the Cretaceous/Tertiary boundary (9–22). In light of these and other studies of ancient biomolecules, paleontological models have proposed that organic biomolecules in ancient samples, such as collagen sequences from the 80 million-year-(my)-old Campanian hadrosaur, Brachylophosaurus canadensis (16) or 68-my-old Tyrannosaurus rex, might be protected by the microenvironment within bones. Such spaces are believed to form a protective shelter that is able to reduce the effects of diagenetic events. In addition to collagen, preserved biomolecules include blood proteins, cellular lipids, and DNA (4, 5). While the maximum estimated lifespan of DNA in bones is ∼20,000 years (ky) at 10 °C, bone proteins have an even longer lifespan, making them an exceptional target for analysis to gain relevant insights into fossilized samples (6). Indeed, the survival of collagen, which is considered to be the most abundant bone protein, is estimated to be in the range 340 ky at 20 °C. Similarly, osteocalcin, the second-most abundant bone protein, can persist for ≈45 ky at 20 °C, thus opening an unprecedented analytical window to study extremely old samples (2, 4, 23).Although ancient DNA amplification and sequencing can yield interesting clues and potential artifacts from contaminating agents (7, 24–28), the improved preservation of ancient proteins provides access to a reservoir of otherwise unavailable genetic information for phylogenetic inference (25, 29, 30). In particular, mass spectrometry (MS)-based screening of species-specific collagen peptides has recently been used as a low-cost, rapid alternative to DNA sequencing for taxonomic attribution of morphologically unidentifiable small bone fragments and teeth stemming from diverse archeological contexts (25, 31–33).For over five decades, researchers have presented biochemical evidence for the existence of preserved protein material from ancient bone samples (34–36). One of the first direct measurements was by amino acid analysis, which showed that the compositional profile of ancient samples was consistent with collagens in modern bone samples (37–39). Preservation of organic biomolecules, either from bone, dentin, antlers, or ivory, has been investigated by radiolabeled ¹⁴C fossil dating (40) to provide an avenue of delineating evolutionary divergence from extant species (3, 41, 42). It is also important to note that these parameters primarily depend on ancient bone collagen as the levels remain largely unchanged (a high percentage of collagen is retained, as gleaned by laboratory experiments on bone taphonomy (6)). Additionally, antibody-based immunostaining methods have given indirect evidence of intact peptide amide bonds (43–45) to aid some of the first evidence of protein other than collagen and osteocalcin in ancient mammoth (43) and human specimens (46).In the past, mass spectrometry has been used to obtain MS signals consistent with modern osteocalcin samples (2, 47), and eventually postsource decay peptide fragmentation was used to confirm the identification of osteocalcin in fossil hominids dating back ∼75 ky (48). More recently, modern “bottom-up” proteomic methods were applied to mastodon and T. rex samples (10), complementing immunohistochemistry evidence (13, 17). The results hinted at the potential of identifying peptides from proteolytic digest of well-preserved bone samples. This work also highlighted the importance of minimizing sources of protein contamination and adhering to data publication guidelines (20, 21). In the past few years, a very well-preserved juvenile mammoth referred to as Lyuba was discovered in the Siberian permafrost and analyzed using high-resolution tandem mass spectrometry (29). This study was followed with a report by Wadsworth and Buckley (30) describing the analysis of proteins from 19 bovine bone samples spanning 4 ky to 1.5 my. Both of these groups reported the identification of additional collagen and noncollagen proteins.Recently, a series of large extinct mammal bones were unearthed at a reservoir near Snowmass Village, Colorado, USA (49, 50). The finding was made during a construction project at the Ziegler Reservoir, a fossil site that was originally a lake formed at an elevation of ∼2,705 m during the Bull Lake glaciations ∼140 ky ago (49, 51). The original lake area was ∼5 hectares in size with a total catchment of ∼14 hectares and lacked a direct water flow inlet or outlet. This closed drainage basin established a relatively unique environment that resulted in the exceptional preservation of plant material, insects (52), and vertebrate bones (49). In particular, a cranial specimen from extinct Bison latifrons was unearthed from the Biostratigraphic Zone/Marine Oxygen Isotope Stage (MIS) 5d, which dates back to ∼120 ky (53, 54).Here, we describe the use of paleoproteomics, for the identification of protein remnants with a focus on a particularly unique B. latifrons cranial specimen found at the Ziegler site. We developed a simplified sample processing approach that allows for analysis of low milligram quantities of ancient samples for peptide identification. Our method avoids the extensive demineralization steps of traditional protocols and utilizes an acid labile detergent to allow for efficient extraction and digestion without the need for additional sample cleanup steps. This approach was applied to a specimen from B. latifrons that displayed visual and mechanical properties consistent with the meninges, a fibrous tissue that lines the cranial cavity. Bioinformatics analysis revealed the presence of a recurring glycosylation signature in well-preserved collagens. In particular, the presence of glycosylated hydroxylysine residues was identified as a unique feature of bone fossil collagen, as gleaned through meta-analyses of raw data from previous reports on woolly mammoth (Mammuthus primigenius) and bovine samples (29, 30). The results from these meta-analyses indicate a common, unique feature of collagen that coincides with, and possibly contributes to its preservation.     

Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Fluorescent labeling of tRNA dihydrouridine residues: Mechanism and distribution

Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH(4), their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNA(Phe) and E. coli tRNA(Arg). The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH(4) reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.     

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.