Bioaerosols in swine confinement buildings: A metaproteomic view.

Swine confinement buildings represent workplaces with high biological air pollution. It is suspected that individual components of inhalable air are causatives of chronic respiratory disease that are regularly detected among workers. In order to understand the relationship between exposure and stress, it is necessary to study the components of bioaerosols in more detail. For this purpose, bioaerosols from pig barns were collected on quartz filters and analysed via a combinatorial approach of 16S rRNA amplicon sequencing and metaproteomics. The study reveals the presence of peptides from pigs, their feed and microorganisms. The proportion of fungal peptides detected is considered to be underrepresented compared to bacterial peptides. In addition, the metaproteomic workflow enabled functional predictions about the discovered peptides. Housekeeping proteins were found in particular, but also evidence for the presence of bacterial virulence factors (e.g., serralysin-like metalloprotease) as well as plant (e.g., chitinase) and fungal allergens (e.g., alt a10). Metaproteomic analyses can thus be used to identify factors that may be relevant to the health of pig farmers. Accordingly, such studies could be used in the future to assess the adverse health potential of an occupationally relevant bioaerosol and help consider defined protective strategies for workers.

Target Mechanisms of the Cyanotoxin Cylindrospermopsin in Immortalized Human Airway Epithelial Cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o-) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.

What Does Ectoine Do to DNA? A Molecular-Scale Picture of Compatible Solute-Biopolymer Interactions.

Compatible solutes accumulate in the cytoplasm of halophilic microorganisms, enabling their survival in a high-salinity environment. Ectoine is such a compatible solute. It is a zwitterionic molecule that strongly interacts with surrounding water molecules and changes the dynamics of the local hydration shell. Ectoine interacts with biomolecules such as lipids, proteins, and DNA. The molecular interaction between ectoine and biomolecules, in particular the interaction between ectoine and DNA, is far from being understood. In this paper, we describe molecular aspects of the interaction between ectoine and double-stranded DNA (dsDNA). Two 20 base pairs-long dsDNA fragments were immobilized on a gold surface via a thiol-tether. The interaction between the dsDNA monolayers with diluted and concentrated ectoine solutions was examined by means of X-ray photoelectron and polarization modulation infrared reflection absorption spectroscopies (PM IRRAS). Experimental results indicate that the ability of ectoine to bind water reduces the strength of hydrogen bonds formed to the ribose-phosphate backbone in the dsDNA. In diluted (0.1 M) ectoine solution, DNA interacts predominantly with water molecules. The sugar-phosphate backbone is involved in the formation of strong hydrogen bonds to water, which, over time, leads to a reorientation of the planes of nucleic acid bases. This reorientation destabilizes the strength of hydrogen bonds between the bases and leads to a partial dehybridization of the dsDNA. In concentrated ectoine solution (2.5 M), almost all water molecules interact with ectoine. Under this condition, ectoine is able to interact directly with DNA. Density functional theory (DFT) calculations demonstrate that the direct interaction involves the nitrogen atoms in ectoine and phosphate groups in the DNA molecule. The results of the quantum-chemical calculations show that rearrangements in the ribose-phosphate backbone, caused by a direct interaction with ectoine, facilitates contacts between the O atom in the phosphate group and H atoms in a nucleic acid base. In the PM IRRA spectra, an increase in the number of IR absorption modes in the base pair frequency region proves that the hydrogen bonds between bases become weaker. Thus, a sequence of reorientations caused by interaction with ectoine leads to a breakdown of hydrogen bonds between bases in the double helix.

Influence of compatible solute ectoine on distinct DNA structures: thermodynamic insights into molecular binding mechanisms and destabilization effects.

In nature, the cellular environment of DNA includes not only water and ions, but also other components and co-solutes, which can exert both stabilizing and destabilizing effects on particular oligonucleotide conformations. Among them, ectoine, known as an important osmoprotectant organic co-solute in a broad range of pharmaceutical products, turns out to be of particular relevance. In this article, we study the influence of ectoine on a short single-stranded DNA fragment and on double-stranded helical B-DNA in aqueous solution by means of atomistic molecular dynamics (MD) simulations in combination with molecular theories of solution. Our results demonstrate a conformation-dependent binding behavior of ectoine, which favors the unfolded state of DNA by a combination of electrostatic and dispersion interactions. In conjunction with the Kirkwood-Buff theory, we introduce a simple framework to compute the influence of ectoine on the DNA melting temperature. Our findings reveal a significant linear decrease of the melting temperature with increasing ectoine concentration, which is found to be in qualitative agreement with results from denaturation experiments. The outcomes of our computer simulations provide a detailed mechanistic rationale for the surprising destabilizing influence of ectoine on distinct DNA structures.

DNA protection by ectoine from ionizing radiation: molecular mechanisms.

Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).

Measurements and simulations of microscopic damage to DNA in water by 30 keV electrons: A general approach applicable to other radiation sources and biological targets.

The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle-scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E_{1/2}=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells.

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations.

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.

Nitrogen fixation in a chemoautotrophic lucinid symbiosis.

The shallow water bivalve Codakia orbicularis lives in symbiotic association with a sulfur-oxidizing bacterium in its gills. The endosymbiont fixes CO2 and thus generates organic carbon compounds, which support the host's growth. To investigate the uncultured symbiont's metabolism and symbiont-host interactions in detail we conducted a proteogenomic analysis of purified bacteria. Unexpectedly, our results reveal a hitherto completely unrecognized feature of the C. orbicularis symbiont's physiology: the symbiont's genome encodes all proteins necessary for biological nitrogen fixation (diazotrophy). Expression of the respective genes under standard ambient conditions was confirmed by proteomics. Nitrogenase activity in the symbiont was also verified by enzyme activity assays. Phylogenetic analysis of the bacterial nitrogenase reductase NifH revealed the symbiont's close relationship to free-living nitrogen-fixing Proteobacteria from the seagrass sediment. The C. orbicularis symbiont, here tentatively named 'Candidatus Thiodiazotropha endolucinida', may thus not only sustain the bivalve's carbon demands. C. orbicularis may also benefit from a steady supply of fixed nitrogen from its symbiont-a scenario that is unprecedented in comparable chemoautotrophic symbioses.

Influence of the Compatible Solute Ectoine on the Local Water Structure: Implications for the Binding of the Protein G5P to DNA.

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-5-protein (G5P) to a single-stranded DNA (dT25). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonucleotide, which has important consequences for osmotic regulation mechanisms.

Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum.

UNLABELLED: Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains are the major cause of colisepticemia (colibacillosis), a condition that has become an increasing public health problem in recent years. ExPEC strains are characterized by high resistance to serum, which is otherwise highly toxic to most bacteria. To understand how these bacteria survive and grow in serum, we performed system-wide analyses of their response to serum, making a clear distinction between the responses to nutritional immunity and innate immunity. Thus, mild heat inactivation of serum destroys the immune complement and abolishes the bactericidal effect of serum (inactive serum), making it possible to examine nutritional immunity. We used a combination of deep RNA sequencing and proteomics in order to characterize ExPEC genes whose expression is affected by the nutritional stress of serum and by the immune complement. The major change in gene expression induced by serum-active and inactive-involved metabolic genes. In particular, the serum metabolic response is coordinated by three transcriptional regulators, Fur, BasR, and CysB. Fur alone was responsible for more than 80% of the serum-induced transcriptional response. Consistent with its role as a major serum response regulator, deletion of Fur renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in colisepticemia and virulence. IMPORTANCE: Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains have emerged as major pathogens, especially in community- and hospital-acquired infections. These bacteria cause a large spectrum of syndromes, the most serious of which is septicemia, a condition with a high mortality rate. These bacterial strains are characterized by high resistance to serum, otherwise highly toxic to most bacteria. To understand the basis of this resistance, we carried out system-wide analyses of the response of ExPEC strains to serum by using proteomics and deep RNA sequencing. The major changes in gene expression induced by exposure to serum involved metabolic genes, not necessarily implicated in relation to virulence. One metabolic regulator-Fur-involved in iron metabolism was responsible for more than 80% of the serum-induced response, and its deletion renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in virulence.

The cognitive capabilities of farm animals: categorisation learning in dwarf goats (Capra hircus).

The ability to establish categories enables organisms to classify stimuli, objects and events by assessing perceptual, associative or rational similarities and provides the basis for higher cognitive processing. The cognitive capabilities of farm animals are receiving increasing attention in applied ethology, a development driven primarily by scientifically based efforts to improve animal welfare. The present study investigated the learning of perceptual categories in Nigerian dwarf goats (Capra hircus) by using an automated learning device installed in the animals' pen. Thirteen group-housed goats were trained in a closed-economy approach to discriminate artificial two-dimensional symbols presented in a four-choice design. The symbols belonged to two categories: category I, black symbols with an open centre (rewarded) and category II, the same symbols but filled black (unrewarded). One symbol from category I and three different symbols from category II were used to define a discrimination problem. After the training of eight problems, the animals were presented with a transfer series containing the training problems interspersed with completely new problems made from new symbols belonging to the same categories. The results clearly demonstrate that dwarf goats are able to form categories based on similarities in the visual appearance of artificial symbols and to generalise across new symbols. However, the goats had difficulties in discriminating specific symbols. It is probable that perceptual problems caused these difficulties. Nevertheless, the present study suggests that goats housed under farming conditions have well-developed cognitive abilities, including learning of open-ended categories. This result could prove beneficial by facilitating animals' adaptation to housing environments that favour their cognitive capabilities.

Early development influences ontogeny of personality types in young laboratory rats.

Features of an individual's early development are frequently reported to alter the postnatal ontogeny in litter-bearing mammals with respect to various physiological parameters. We hypothesized that such effects might also apply to the ontogeny of personality types. On the one hand, litter size effects by means of more contacts with littermates, might lead to the development of more offensive types. On the other hand, smaller and less developed young from larger litters might be less offensive due to their lower physical capabilities to deal with challenging situations. We studied these contrasting hypotheses in young rats, which we tested in a battery of emotionality tests. There were clear indications for the existence of distinct behavioral types by means of consistencies in behavioral responses within and across contexts. Based on these responses, we calculated three new variables by PCA, which we interpreted to mainly reflect boldness, exploration, and anxiety. Overall, our results strongly suggest that the early development alters the ontogeny of personality types, with heavier individuals being bolder and more explorative. Furthermore, body mass and litter size influenced the changes in the behavioral responses in successive tests, further supporting the importance of the litter size-dependent body mass for the ontogeny of personalities. Anxiety also depended on litter size, however, in a nonlinear way. Animals born to litters of small or large sizes had higher scores, whereas individuals from medium-sized litters were less anxious. This optimum curve indicates that opposing effects of litter size are involved in shaping personalities in young rats.

[Cognitive enrichment in zoo and farm animals--implications for animal behaviour and welfare]. / Kognitive Umweltanreicherung bei Zoo- und Nutztieren--Implikationen für Verhalten und Wohlbefinden der Tiere.

Animals in the wild are facing a wide variety of challenges and ever-changing environmental stimuli. For successful coping, animals use both innate behavioural programs and their cognitive skills. In contrast, zoo- and farm animals have to cope with restricted husbandry conditions, which offer only few opportunities to adequately satisfy their various needs. Consequences could be sensory and cognitive underchallenge that can cause boredom and frustration as well as behavioural disturbances. Initially intended for improvement of management and husbandry, different forms of operant behavioural training have been applied firstly in zoo- and later also in farm animals. It has been suggested that successful coping with appropriate cognitive challenges is a source of positive emotions and may lead to improved welfare. Under the term cognitive enrichment, new approaches have been developed to integrate cognitive challenges into the housing of zoo- and farm animals. The present article reviews actual research in the field. Previous results indicate that, beyond improvement of management and handling routines, such approaches can positively affect animal behaviour and welfare. The combination of explorative and appetitive behaviour with successful learning improves environmental predictability and controllability for the animals, activates reward-related brain systems and can directly affect emotional processes of appraisal. For practical implementation in farm animal husbandry, it sounds promising to link individual access to e.g. automated feeders or milking systems with previously conditioned stimuli and/or discriminatory learning tasks. First experimental approaches in pigs, dwarf goats and cattle are available and will be discussed in the present article.

Litter size is negatively correlated with corticosterone levels in weanling and juvenile laboratory rats.

An animal's environment during early life can strongly affect its physiological development. For example, litter size, i.e. the number of litter siblings, has been previously shown to strongly affect early growth in many small mammal species including laboratory rats. In the present study we tested whether natural, unmanipulated litter size is also associated with differences in stress hormone levels in young Long-Evans laboratory rats. We found a negative correlation between serum corticosterone (CORT) concentrations and litter size during two different stages of juvenile life. On postnatal day 17, shortly before weaning, this relationship was apparent with respect to basal CORT values. On day 33, however, two weeks after weaning, we found this relationship only when animals were challenged by a 10-min test on an elevated plus maze, but not in control animals (basal values). Although the physiological basis of these differences is not clear, we discuss two main, not mutually exclusive possibilities: (a) delayed maturation of the HPA axis in typically lower body mass pups of large litters, and (b) that such pups, encountering greater competition for maternal resources, adjust to this presumably more stressful developmental environment by down-regulating responsiveness of the HPA axis. In conclusion, our study provides evidence that a naturally varying feature of the developmental environment of many altricial mammals - number of littermates - may contribute to individual differences in stress-related physiology. Furthermore, it suggests the need to consider litter-size effects when investigating differences in animals' stress responses.