The virtual ecologist approach: simulating data and observers

Ecologists carry a well‐stocked toolbox with a great variety of sampling methods, statistical analyses and modelling tools, and new methods are constantly appearing. Evaluation and optimisation of these methods is crucial to guide methodological choices. Simulating error‐free data or taking high‐quality data to qualify methods is common practice. Here, we emphasise the methodology of the ‘virtual ecologist’ (VE) approach where simulated data and observer models are used to mimic real species and how they are ‘virtually’ observed. This virtual data is then subjected to statistical analyses and modelling, and the results are evaluated against the ‘true’ simulated data. The VE approach is an intuitive and powerful evaluation framework that allows a quality assessment of sampling protocols, analyses and modelling tools. It works under controlled conditions as well as under consideration of confounding factors such as animal movement and biased observer behaviour. In this review, we promote the approach as a rigorous research tool, and demonstrate its capabilities and practical relevance. We explore past uses of VE in different ecological research fields, where it mainly has been used to test and improve sampling regimes as well as for testing and comparing models, for example species distribution models. We discuss its benefits as well as potential limitations, and provide some practical considerations for designing VE studies. Finally, research fields are identified for which the approach could be useful in the future. We conclude that VE could foster the integration of theoretical and empirical work and stimulate work that goes far beyond sampling methods, leading to new questions, theories, and better mechanistic understanding of ecological systems.     

The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.In eukaryotes, the ubiquitous second messenger cyclic AMP (cAMP) is generated from ATP by membrane-integral or by cytoplasmic, CO₂-regulated cyclases (35, 44). The cAMP signal is processed by a small group of receiver proteins, including the regulatory subunit of protein kinase A (28), cAMP-gated ion channels (4), and the guanine-nucleotide-exchange proteins EPAC1 and EPAC2 (39). The cAMP signal is terminated by the action of a family of cyclic nucleotide-specific phosphodiesterases (PDEs) (9). This paradigm is rather straightforward, involves a limited number of players, and is generally well understood, at least in mammalian cells. However, much less is known about how individual cAMP signals are temporally and spatially controlled. Since most eukaryotic adenylyl cyclases are integral membrane proteins, often restricted to specific membrane subdomains (10), cAMP signaling is usually initiated at the cell membrane (40). However, diffusion of cAMP away from its site of generation is rapid, with diffusion coefficients being about 400 μm²/s (8, 15, 29), translating into diffusion velocities of 30 to 40 μm/s. As a consequence, the signal would reach the center of the cell with a diameter of 3 μm within less than 50 ms and would rapidly saturate the entire cell. While regulation through fluctuating cellular levels of cAMP represents a valid paradigm of cAMP signaling, it has become clear that other, more localized modes of cAMP signaling must also exist. Several groups have shown that the cAMP response of a given cell can differ depending on what set of receptors activates the cyclase response (14, 30, 41, 42). Similarly, the cAMP response of endothelial cells depends on the subcellular site where the cAMP is produced. They tighten their barrier function when cAMP is produced by membrane-bound adenylyl cyclases but become more permeable when cAMP is produced in the cytoplasm (17, 45). The distinct subcellular localization of cAMP signals was experimentally demonstrated using an array of techniques (29, 40, 55, 56).Physically tethered PDEs might serve to confine newly synthesized cAMP to defined microdomains. Only cAMP-binding proteins that are localized within or extend into such microdomains would be able to receive the cAMP signal (17, 49). cAMP concentrations within such domains might rise and fall rapidly, reaching peak concentrations much more rapidly and locally far beyond the steady-state cAMP levels measured in whole-cell extracts. Such spatially organized, tethered PDEs can generate local sinks into which cAMP disappears (1, 23). This paradigm would allow the simultaneous presence of numerous local cAMP concentration gradients within a single cell, allowing great flexibility in signal generation and intracellular signal transmission. This concept is based on the distinct subcellular localization and physical association of PDEs with subcellular structures and on the existence of localized subcellular cAMP pools, for which there is extensive experimental support (3, 5, 13, 50, 52). Interestingly, PDEs localized in different subcellular regions may still be able to compensate for each other. Ablation of the cilium-specific PDE1C from the olfactory neurons in the mouse did not prolong response termination, as long as the cytoplasmic PDE4 in the cell body was still present (11).The unicellular eukaryote Trypanosoma brucei is the causative agent of human sleeping sickness in sub-Saharan Africa. It belongs to the large order of the kinetoplastida, which includes many medically and economically important pathogens of humans, their livestock, and their crops worldwide (27). Trypanosomes are very small cells (about 15 by 3 μm in diameter) that carry a single flagellum (10 by 0.5 μm). The volume of a procyclic trypanosome of strain 427 is (9.6 ± 0.8) × 10⁻¹⁴ liter (Markus Engstler, personal communication), with the flagellum representing about 15% of this. A signaling threshold concentration of 1 μM cAMP corresponds to just about 30,000 molecules of cAMP per cell. Given a diffusion coefficient of 400 μm²/s (29), unrestricted diffusion of cAMP would swamp the cell within 50 ms. Obviously, temporal and spatial control of cAMP signaling is crucial for T. brucei. Strategically located, physically tethered PDEs might thus play an important role in the architecture of the cAMP signaling pathways in T. brucei.The genomes of T. brucei and of other kinetoplastids, such as T. vivax, T. cruzi, Leishmania major, L. infantum, and L. braziliensis, all code for the same set of five cyclic nucleotide-specific PDEs (25, 53). In T. brucei, the genes for T. brucei PDEB1 (TbrPDEB1; subsequently termed PDEB1) and TbrPDEB2 (PDEB2) are tandemly arranged on chromosome 9 and code for two very similar cAMP-specific PDEs, each with two GAF (mammalian cyclic GMP-dependent PDEs, Anabaena adenylyl cyclases, Escherichia coli FhlA) domains (21) in their N-terminal regions (38, 57). These two PDEs were also studied experimentally in T. cruzi (12) and L. major (24, 52), and orthologues are present in all kinetoplastid genomes available so far. Despite their high overall sequence similarity, PDEB1 and PDEB2 exhibit distinct subcellular localizations (31). PDEB1 is predominantly found in the flagellum, where it is stably associated with cytoskeletal components that are resistant to detergent extraction. In contrast, PDEB2 is mostly localized in the cell body, from where it is fully extractable by nonionic detergents. However, a minor fraction of PDEB2 also associates with the flagellar skeleton in a Triton-resistant manner, most likely through interaction with PDEB1. Earlier work has shown that both PDEB1and PDEB2 are essential enzymes in bloodstream-form T. brucei (31), while TbPDEA, TbPDEC, and TbPDED play minor roles (20; S. Kunz, unpublished data).     

Treatment of chronic endometritis in dairy cows with an intrauterine application of enzymes. A field trial

The use of proteolytic enzymes has been established in the non-antibiotic treatment of mastitis in dairy cattle. The objective of this study was to evaluate, if enzymes are efficacious in the treatment of chronic endometritis. In a controlled field trial, cows with vaginal discharge 21-27 days in milk (DIM) were randomly assigned to two treatment groups. Endometritis was classified into three categories, depending on the type of vaginal discharge: clear mucus with flakes of pus (E1), mucopurulent discharge or fluctuating contents in the uterus (E2), and purulent discharge (E3). In group ENZYMES (n=191), cows received an intrauterine treatment with a salve containing the enzymes trypsin (16 mg), chymotrypsin (16 mg), and papain (8 mg). Cows in group PGF (n=225) were treated with 0.5mg of cloprostenol. Cows that did not show any clinical signs of chronic endometritis were regarded as healthy control group (HC, n=699). In groups ENZYMES and PGF, all cows were re-examined 35-41 DIM. In group ENZYMES, cows were re-treated with enzymes if signs of endometritis were found, while in group PGF all cows received a second dose of cloprostenol, regardless of their clinical findings. Cure rate after the first treatment, defined as the absence of vaginal discharge at the re-examinations, was 59.7 and 68.0% in groups ENZYMES and PGF, respectively (P>0.05). Reproductive performance measures showed no significant differences between the two treatment groups. Service rate was significantly lower for ENZYMES and PGF, respectively, compared to HC. Conception rates to all services and percentages of cows pregnant by 250 DIM were significantly lower in group ENZYMES compared to HC, while no further differences were found between PGF and HC. In both treatment groups, cure rate and reproductive performance measures were better for cows categorized E1 or E2, than for cows categorized E3, respectively. Conception rate to all services for cows with endometritis category E1 was higher in group PGF than in group ENZYMES (P<0.05). The results of this field trial suggest that prostaglandin F(2alpha) is still the treatment of choice for chronic endometritis in dairy cattle.     

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.     

On the way from morphology to phonology: German linking elements and the role of the phonological word

German linking elements are sometimes classified as inflectional affixes, sometimes as derivational affixes, and in any case as morphological units with at least seven realisations (e.g. -s-, -es-, -(e)n-, -e-). This article seeks to show that linking elements are hybrid elements situated between morphology and phonology. On the one hand, they have a clear morphological status since they occur only within compounds (and before a very small set of suffixes) and support the listener in decoding them. On the other hand, they also have to be analysed on the phonological level, as will be shown in this article. Thus, they are marginal morphological units on the pathway to phonology (including prosodics). Although some alloforms can sometimes be considered former inflectional endings and in some cases even continue to demonstrate some inflectional behaviour (such as relatedness to gender and inflection class), they are on their way to becoming markers of ill-formed phonological words. In fact, linking elements, above all the linking -s-, which is extremely productive, help the listener decode compounds containing a bad phonological word as their first constituent, such as Geburt+s+tag ‘birthday’ or Religion+s+unterricht ‘religious education’. By marking the end of a first constituent that differs from an unmarked monopedal phonological word, the linking element aids the listener in correctly decoding and analysing the compound. German compounds are known for their length and complexity, both of which have increased over time—along with the occurrence of linking elements, especially -s-. Thus, a profound instance of language change can be observed in contemporary German, one indicating its typological shift from syllable language to word language.     

Macroecology in the age of Big Data – Where to go from here?

Recent years have seen an exponential increase in the amount of data available in all sciences and application domains. Macroecology is part of this “Big Data” trend, with a strong rise in the volume of data that we are using for our research. Here, we summarize the most recent developments in macroecology in the age of Big Data that were presented at the 2018 annual meeting of the Specialist Group Macroecology of the Ecological Society of Germany, Austria and Switzerland (GfÖ). Supported by computational advances, macroecology has been a rapidly developing field over recent years. Our meeting highlighted important avenues for further progress in terms of standardized data collection, data integration, method development and process integration. In particular, we focus on (a) important data gaps and new initiatives to close them, for example through space- and airborne sensors, (b) how various data sources and types can be integrated, (c) how uncertainty can be assessed in data-driven analyses and (d) how Big Data and machine learning approaches have opened new ways of investigating processes rather than simply describing patterns. We discuss how Big Data opens up new opportunities, but also poses new challenges to macroecological research. In the future, it will be essential to carefully assess data quality, the reproducibility of data compilation and analytical methods, and the communication of uncertainties. Major progress in the field will depend on the definition of data standards and workflows for macroecology, such that scientific quality and integrity are guaranteed, and collaboration in research projects is made easier.