Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons

For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).     

Comparison of cellular and humoral immunoassays for the assessment of summer eczema in horses

The objective of this study was to compare and analyze three common diagnostic methods for summer eczema (SE) in horses, an allergic dermatitis caused by bites of Culicoides spp. Nine horses with a medical history of SE and nine control animals were intradermally challenged with whole body extracts (WBE) and the saliva of a native (C. nubeculosus) and exotic (C. sonorensis) Culicoides species. Blood and serum samples of the horses were examined for basophil reactivity by a histamine release test (HRT) and for Culicoides-specific serum immunoglobulin E (IgE) and G (IgG) by enzyme-linked immunosorbent assay (ELISA). The results of intradermal testing (IDT) at 30min (immediate reactivity) and 4h (late-phase reactivity) post challenge with most insect preparations revealed significant differences between horses with and without SE. Overall, the HRT showed the most accurate results with a sensitivity of 1.00 for all Culicoides preparations and specificities of 0.78 (WBE) and 1.00 (saliva). By contrast, delayed reactions of the IDT (24h), and levels of Culicoides-specific IgE and IgG in the native serum showed little or no distinction between allergic and non-allergic horses. However, the use of purified serum IgE and IgG indicated the possibility for elevated titers of insect-specific serum immunoglobulins in horses with SE. The IDT and HRT did not reveal obvious differences in onset and intensity of positive reactions for the native verses exotic Culicoides species, whereas the ELISA showed slightly higher numbers of positive reactions for serum IgG with the indigenous species. Saliva, as compared to WBE, was found to have improved sensitivity and/or specificity for the HRT and for the late-phase immune reactions as measured by the IDT. Overall, the results indicate that allergy tests utilizing effector cells (mast cells, basophils) are more accurate in diagnosing SE in horses than serological analysis by ELISA.     

Perception and acceptance of agricultural production in and on urban buildings (ZFarming): a qualitative study from Berlin,Germany

Rooftop gardens, rooftop greenhouses and indoor farms (defined as ZFarming) have been established or planned by activists and private companies in Berlin. These projects promise to produce a range of goods that could have positive impacts on the urban setting but also carry a number of risks and uncertainties. In this early innovation phase, the relevant stakeholders’ perceptions and social acceptance of ZFarming represent important preconditions for success or failure of the further diffusion of this practice. We used the framework of acceptance to investigate the stakeholders’ attitudes and to identify the key factors that might hinder or promote the introduction of ZFarming. The results are based on an analysis of 38 qualitative interviews conducted with key stakeholders in Berlin. As the results show, major perceived benefits of ZFarming include improved consumer awareness, education, and the creation of experimental spaces. Stakeholders further perceive opportunities for resource savings, new business models, repurposing of abandoned buildings and improved aesthetics. Major perceived risks are associated with growing techniques that are considered “unnatural”, health risks (due to urban pollutants), conflicts with images of traditional agriculture, the rejection of animal production in urban areas, the risk of projects being too expensive and too complex or being implemented too early, i.e. before the mechanisms are fully understood. The analysis further reveals which contextual factors—political, legal, market-related, spatial or societal—might negatively or positively influence ZFarming acceptance.     

Histopathological findings,phenotyping of inflammatory cells,and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

Background

The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.

Results

The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.

Conclusions

The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.     

Applying Bayesian network modelling to understand the links between on-farm biosecurity practice during the 2007 equine influenza outbreak and horse managers’ perceptions of a subsequent outbreak

Australia experienced its first ever outbreak of equine influenza in August 2007. Horses on 9359 premises were infected over a period of 5 months before the disease was successfully eradicated through the combination of horse movement controls, on-farm biosecurity and vaccination. In a previous premises-level case-control study of the 2007 equine influenza outbreak in Australia, the protective effect of several variables representing on-farm biosecurity practices were identified. Separately, factors associated with horse managers’ perceptions of the effectiveness of biosecurity measures have been identified.     

Mineralization of carbon and nitrogen from cowpea leaves decomposing in soils with different levels of microbial biomass

Soils with greater levels of microbial biomass may be able to release nutrients more rapidly from applied plant material. We tested the hypothesis that the indigenous soil microbial biomass affects the rate of decomposition of added green manure. Cowpea (Vigna unguiculata L.) Walp.] leaves were added to four soils with widely differing microbial biomass C levels. C and N mineralization of the added plant material was followed during incubation at 30°C for 60 days. Low levels of soil microbial biomass resulted in an initially slower rate of decomposition of soil-incorporated green manure. The microbial biomass appeared to adjust rapidly to the new substrate, so that at 60 days of incubation the cumulative C loss and net N mineralization from decomposing cowpea leaves were not significantly affected by the level of the indigenous soil microbial biomass.     

Rabies in a 10-week-old puppy

A 10-week-old, male, border collie-cross puppy was examined for an acute onset of unilateral vestibular signs. Neurologic deterioration was rapid over the next 12 hours and the puppy was euthanized. Rabies was diagnosed by histopathologic and immunohistochemical examination.