High Fat,Low Carbohydrate Diet Limit Fear and Aggression in G?ttingen Minipigs

High fat, low carbohydrate diets have become popular, as short-term studies show that such diets are effective for reducing body weight, and lowering the risk of diabetes and cardiovascular disease. There is growing evidence from both humans and other animals that diet affects behaviour and intake of fat has been linked, positively and negatively, with traits such as exploration, social interaction, anxiety and fear. Animal models with high translational value can help provide relevant and important information in elucidating potential effects of high fat, low carbohydrate diets on human behaviour. Twenty four young, male Göttingen minipigs were fed either a high fat/cholesterol, low carbohydrate diet or a low fat, high carbohydrate/sucrose diet in contrast to a standard low fat, high carbohydrate minipig diet. Spontaneous behaviour was observed through video recordings of home pens and test-related behaviours were recorded during tests involving animal-human contact and reaction towards a novel object. We showed that the minipigs fed a high fat/cholesterol, low carbohydrate diet were less aggressive, showed more non-agonistic social contact and had fewer and less severe skin lesions and were less fearful of a novel object than minipigs fed low fat, high carbohydrate diets. These results found in a porcine model could have important implications for general health and wellbeing of humans and show the potential for using dietary manipulations to reduce aggression in human society.     

Virus and Autoantigen-Specific CD4+ T Cells Are Key Effectors in a SCID Mouse Model of EBV-Associated Post-Transplant Lymphoproliferative Disorders

Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies.     

Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.     

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.     

A Novel Staining Protocol for Multiparameter Assessment of Cell Heterogeneity in Phormidium Populations (Cyanobacteria) Employing Fluorescent Dyes

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, ‘dead cell’ nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead.     

Co-invasion of three Asian earthworms, <Emphasis Type="Italic">Metaphire hilgendorfi</Emphasis>, <Emphasis Type="Italic">Amynthas agrestis</Emphasis> and <Emphasis Type="Italic">Amynthas tokioensis</Emphasis> in the USA

Earthworm invasions are one of the most serious causes of ecological deterioration in the temperate deciduous forests of North America. Non-native earthworms impact understory vegetation, leaf litter layer, carbon dynamics, nutrient availability, and the associated food webs. Here we report a significant status change and confirm expansion of known range of Amynthas agrestis, one of the most serious invasive species in North America, and two of its close relatives, A. tokioensis and Metaphire hilgendorfi. The three species have never been confirmed to co-occur in North American ecosystems. We examined 1760 earthworms collected from 30 sites across northeastern USA, and identified them using a new morphological key. Our data show that sympatric occurrence of at least two, and often all three, species is more common than having only one species. In addition, A. tokioensis was dominant in many of these earthworm communities. The status change in species composition from only one species to two or three and the shift in dominance are most likely caused by previous incorrect species identification. Our results support expansion of known range of A. tokioensis and M. hilgendorfi northward and westward into states with colder winters. This range expansion may have taken place alongside that of A. agrestis in the last 10–20 years, but has long been overlooked. Altogether, results highlight an urgent need for correct species identification. The recognition of an expanding multi-species system represents a unique opportunity to further evaluate complex interactions among co-invading and resident species, and to investigate whether interspecific interactions have unexpected non-additive impacts on ecological processes.     

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time.The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways.Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.     

Directional specificity and encoding of muscle forces and loads by stick insect tibial campaniform sensilla,including receptors with round cuticular caps

In many systems, loads are detected as the resistance to muscle contractions. We studied responses to loads and muscle forces in stick insect tibial campaniform sensilla, including a subgroup of receptors (Group 6B) with unusual round cuticular caps in oval-shaped collars. Loads were applied in different directions and muscle contractions were emulated by applying forces to the tibial flexor muscle tendon (apodeme). All sensilla 1) were maximally sensitive to loads applied in the plane of joint movement and 2) encoded muscle forces but did not discharge to unresisted movements. Identification of 6B sensilla by stimulation of cuticular caps demonstrated that receptor responses were correlated with their morphology. Sensilla with small cuticular collars produced small extracellular potentials, had low thresholds and strong tonic sensitivities that saturated at moderate levels. These receptors could effectively signal sustained loads. The largest spikes, derived from sensilla with large cuticular collars, had strong dynamic sensitivities and signaled a wide range of muscle forces and loads. Tibial sensilla are apparently tuned to produce no responses to inertial forces, as occur in the swing phase of walking. This conclusion is supported by tests in which animals 'stepped' on a compliant surface and sensory discharges only occurred in stance.     

Mechanisms underpinning community stability along a latitudinal gradient: Insights from a niche-based approach

At large scales, the mechanisms underpinning stability in natural communities may vary in importance due to changes in species composition, mean abundance, and species richness. Here we link species characteristics (niche positions) and community characteristics (richness and abundance) to evaluate the importance of stability mechanisms in 156 butterfly communities monitored across three European countries and spanning five bioclimatic regions. We construct niche-based hierarchical structural Bayesian models to explain first differences in abundance, population stability, and species richness between the countries, and then explore how these factors impact community stability both directly and indirectly (via synchrony and population stability). Species richness was partially explained by the position of a site relative to the niches of the species pool, and species near the centre of their niche had higher average population stability. The differences in mean abundance, population stability, and species richness then influenced how much variation in community stability they explained across the countries. We found, using variance partitioning, that community stability in Finnish communities was most influenced by community abundance, whereas this aspect was unimportant in Spain with species synchrony explaining most variation; the UK was somewhat intermediate with both factors explaining variation. Across all countries, the diversity–stability relationship was indirect with species richness reducing synchrony which increased community stability, with no direct effects of species richness. Our results suggest that in natural communities, biogeographical variation observed in key drivers of stability, such as population abundance and species richness, leads to community stability being limited by different factors and that this can partially be explained due to the niche characteristics of the European butterfly assemblage.     

Cyanobacteria inhabiting biological soil crusts of a polar desert: Sør Rondane Mountains,Antarctica

Molecular and morphological methods were applied to study cyanobacterial community composition in biological soil crusts (BSCs) from four areas (two nunataks and two ridges) in the Sør Rondane Mountains, Antarctica. The sampling sites serve as control areas for open top chambers (OTCs) that were put in place in 2010 at the time of sample collection and will be compared with BSC samples taken from the OTCs in the future. Cyanobacterial cell biovolume was estimated using epifluorescence microscopy, which revealed the dominance of filamentous cyanobacteria in all studied sites except the Utsteinen ridge, where unicellular cyanobacteria were the most abundant. Cyanobacterial diversity was studied by a combination of molecular fingerprinting methods based on the 16S rRNA gene (denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing) using cyanobacteria-specific primers. The number of DGGE sequences obtained per site was variable and, therefore, a high-throughput method was subsequently employed to improve the diversity coverage. Consistent with previous surveys in Antarctica, both methods showed that filamentous cyanobacteria, such as Leptolyngbya sp., Phormidium sp. and Microcoleus sp., were dominant in the studied sites.In addition, the studied localities differed in substrate type, climatic conditions and soil parameters, which probably resulted in differences in cyanobacterial community composition. Furthermore, the BSC growing on gneiss pebbles had lower cyanobacterial abundances than BSCs associated with granitic substrates.