Process‐based long‐term evaluation of an ecological network of calcareous grasslands connected by sheep herding

This paper evaluates the long‐term effect of an ecological network of calcareous grasslands, a habitat type that experienced dramatic habitat loss and fragmentation during the 20th century, on species richness of habitat specialist plants. Calcareous grasslands are of special conservation concern as the habitat type with the highest diversity in plant and invertebrate species in central Europe. A baseline survey in 1989 established complete vascular plant species lists for all 62 previously abandoned calcareous grassland patches in the study area and assessed the presence of 48 habitat specialist plant species. An ecological network was initiated in 1989 to reconnect these patches with existing grazed pastures (core areas) through large flock sheep herding where feasible, as sheep are thought to be the primary dispersal vectors for calcareous grassland plants. An evaluation survey in 2009 showed significant increase in species richness of habitat specialist plants in patches reconnected by sheep herding, indicating successful colonizations by habitat specialist plants, while ungrazed patches showed no significant change. Observed increase in species richness between 1989 and 2009 was related to connectivity by sheep herding and the presence of a diversity of structural elements providing microsites for establishment. Baseline species richness of the patches, which had been abandoned since at least 1960, was associated with patch area, supporting the effect of ecological drift, and with vegetation type, which suggests that delays in extinction may be related to site factors governing the strength of competition with later seral species. The implementation of this ecological network represents a long‐term ‘natural experiment’ with baseline data, manipulation, and evaluation of hypothesized effects on a clearly defined target variable. It thus provides much needed empirical evidence that species loss in fragmented calcareous grassland communities can be counteracted by restoring functional connectivity among remnant patches.     

Phenotypic plasticity closely linked to climate at origin and resulting in increased mortality under warming and frost stress in a common grass

Phenotypic plasticity is important for species responses to global change and species coexistence. Phenotypic plasticity differs among species and traits and changes across environments. Here, we investigated phenotypic plasticity of the widespread grass Arrhenatherum elatius in response to winter warming and frost stress by comparing phenotypic plasticity of 11 geographically and environmentally distinct populations of this species to phenotypic plasticity of populations of different species originating from a single environment. The variation in phenotypic plasticity was similar for populations of a single species from different locations compared to populations of functionally and taxonomically diverse species from one environment for the studied traits (leaf biomass production and root integrity after frost) across three indices of phenotypic plasticity (RDPI, PIN, slope of reaction norm). Phenotypic plasticity was not associated with neutral genetic diversity but closely linked to the climate of the populations’ origin. Populations originating from warmer and more variable climates showed higher phenotypic plasticity. This indicates that phenotypic plasticity can itself be considered as a trait subject to local adaptation to climate. Finally, our data emphasize that high phenotypic plasticity is not per se positive for adaptation to climate change, as differences in stress responses are resulting in high phenotypic plasticity as expressed by common plasticity indices, which is likely to be related to increased mortality under stress in more plastic populations.     

Introducing “best single template” models as reference baseline for the Continuous Automated Model Evaluation (CAMEO)

Critical blind assessment of structure prediction techniques is crucial for the scientific community to establish the state of the art, identify bottlenecks, and guide future developments. In Critical Assessment of Techniques in Structure Prediction (CASP), human experts assess the performance of participating methods in relation to the difficulty of the prediction task in a biennial experiment on approximately 100 targets. Yet, the development of automated computational modeling methods requires more frequent evaluation cycles and larger sets of data. The “Continuous Automated Model EvaluatiOn (CAMEO)” platform complements CASP by conducting fully automated blind prediction evaluations based on the weekly pre-release of sequences of those structures, which are going to be published in the next release of the Protein Data Bank (PDB). Each week, CAMEO publishes benchmarking results for predictions corresponding to a set of about 20 targets collected during a 4-day prediction window. CAMEO benchmarking data are generated consistently for all methods at the same point in time, enabling developers to cross-validate their method's performance, and referring to their results in publications. Many successful participants of CASP have used CAMEO—either by directly benchmarking their methods within the system or by comparing their own performance to CAMEO reference data. CAMEO offers a variety of scores reflecting different aspects of structure modeling, for example, binding site accuracy, homo-oligomer interface quality, or accuracy of local model confidence estimates. By introducing the "bestSingleTemplate" method based on structure superpositions as a reference for the accuracy of 3D modeling predictions, CAMEO facilitates objective comparison of techniques and fosters the development of advanced methods.     

A new in vitro bioassay for cyst formation by renal cells from an autosomal dominant rat model of polycystic kidney disease

Summary Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent human inherited diseases. The main feature of the disease is the development of renal cysts, first occurring in the proximal tubules, and with time, dominating all segments of the nephron, leading to end-stage renal disease in 50% of the patients in their fifth decade of life. A therapy for polycystic kidney disease (PKD) has not yet been developed. Patients coming to end-stage ADPKD require long-term dialysis and/or transplantation. A suitable animal model to study ADPKD is the spontaneously mutated Han:SPRD (cy/ +) rat, but a method to cultivate Han:SPRD (cy/ +) derived renal cells which preserves their ability to form cyst-like structures in vitro has previously not been reported. Based on this well-characterized animal model, we developed a cell culture model of renal cyst formation in vitro. When renal cells of the Han:SPRD (cy/ +) rat were isolated and cultured under conditions that prevent cell-substratum adhesion, large amounts of cyst-like structures were formed de novo from Han:SPRD (cy/ +) derived renal cells, but only a few from control rat renal cells. In contrast, when cultivated on plastic as monolayer cultures, Han:SPRD (cy/ +)-derived and control rat-derived renal cells were indistinguishable and did not form cyst-like structures. Immunohistochemical characterization of the cyst-like structures suggests tubular epithelial origin of the cyst-forming cells. The amount of cysts formed from Han:SPRD (cy/ +)-derived renal cells grown in a stationary suspension culture is susceptible to modulation by different conditions. Human cyst fluid and epidermal growth factor both stimulated the formation of cysts from Han:SPRD (cy/ +)-derived renal cells whereas taxol inhibited cystogenesis. In contrast, neither human cyst fluid nor epidermal growth factor affected the amount of cysts formed by control rat renal cells. As the culture model reported here allows not only the distinction of PKD-derived tubular epithelium from its normal counterpart, but also the modulation of cyst formation especially by Han:SPRD (cy/ +)-derived renal cells, it might be a useful prescreening protocol for potential treatments for PKD and thus reduce the need for animal experiments. Both authors contributed equally to the work.     

Fiber optic in vivo imaging in the mammalian nervous system

The compact size, mechanical flexibility, and growing functionality of optical fiber and fiber optic devices are enabling several new modalities for imaging the mammalian nervous system in vivo. Fluorescence microendoscopy is a minimally invasive fiber modality that provides cellular resolution in deep brain areas. Diffuse optical tomography is a non-invasive modality that uses assemblies of fiber optic emitters and detectors on the cranium for volumetric imaging of brain activation. Optical coherence tomography is a sensitive interferometric imaging technique that can be implemented in a variety of fiber based formats and that might allow intrinsic optical detection of brain activity at a high resolution. Miniaturized fiber optic microscopy permits cellular level imaging in the brains of behaving animals. Together, these modalities will enable new uses of imaging in the intact nervous system for both research and clinical applications.     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.