An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying stability among three spore types

Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.     

Effects of serial subculture in vitro on the endogenous levels of indole-3-acetic acid and abscisic acid and rootability in microcuttings of ‘Jonathan’ apple

Proliferating axillary shoots of the difficult-to-root apple cultivar Jonathan acquired an enhanced ability to form adventitious roots with increasing number of subcultures in vitro. The transition between the difficult-to-root and the easy-to-root condition occurred at the fourth subculture.Endogenous levels of free IAA and ABA in shoot tissues were analysed by gas chromatography/mass spectrometry/single ion monitoring (GC/MS/SIM) using negative ion chemical ionisation. Tissues from the mother plants grown in the glasshouse contained more IAA and ABA than those from tissue-culture material. After establishment in vitro there was no variation in the IAA content throughout the subcultures but a decrease in ABA content was observed after the fourth transfer. The IAA/ABA ratio increased from 0.2 in difficult-to-root shoots from the initial culture up to 0.7 in easy-to-root shoots from the long-term subculture.     

Tuning the white light spectrum of light emitting diode lamps to reduce attraction of nocturnal arthropods

Artificial lighting allows humans to be active at night, but has many unintended consequences, including interference with ecological processes, disruption of circadian rhythms and increased exposure to insect vectors of diseases. Although ultraviolet and blue light are usually most attractive to arthropods, degree of attraction varies among orders. With a focus on future indoor lighting applications, we manipulated the spectrum of white lamps to investigate the influence of spectral composition on number of arthropods attracted. We compared numbers of arthropods captured at three customizable light-emitting diode (LED) lamps (3510, 2704 and 2728 K), two commercial LED lamps (2700 K), two commercial compact fluorescent lamps (CFLs; 2700 K) and a control. We configured the three custom LEDs to minimize invertebrate attraction based on published attraction curves for honeybees and moths. Lamps were placed with pan traps at an urban and two rural study sites in Los Angeles, California. For all invertebrate orders combined, our custom LED configurations were less attractive than the commercial LED lamps or CFLs of similar colour temperatures. Thus, adjusting spectral composition of white light to minimize attracting nocturnal arthropods is feasible; not all lights with the same colour temperature are equally attractive to arthropods.     

Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly.     

Crystal Structure of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-Å resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S]²⁺) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr⁶⁰, Trp²⁶⁴, and Trp³⁵⁵ provide the framework for the “aromatic bowl” that serves as a trimethylamine-binding site in TMADH is comprised of Gln⁶⁵, Trp²⁶⁷, and Asp³⁵⁸, respectively, in HADH. The surface Tyr⁴⁴² that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.     

The toxoplasma proteins MIC2 and M2AP form a hexameric complex necessary for intracellular survival

Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2 x M2AP complex participates in host cell attachment and invasion. The short cytoplasmic C-domain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2 x M2AP complex of approximately 450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three alphabeta dimers. Resolution of this complex has implications for how MIC2 x M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.     

Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents.