Strain identification inRhizobium meliloti using the antibiotic disk susceptibility test

Summary The antibiotic disk susceptibility test was used to measure the variation in the intrinsic resistance of 49 strains ofRhizobium meliloti to 9 antibiotics. Several strains had unique patterns of resistance. However, during cluster analysis, when a minimum Euclidean distance equal to 4 was used as a discriminating tool, the strains were grouped in 12 groups. The largest group contained 74% of the strains but 9 strains (2 very effective, 4 effective and 3 ineffective) showed very unique patterns of resistance and formed 9 distinct groups.R. meliloti strains in general showed high intrinsic resistance to the 9 antibiotics tested.Contribution no. 189 Station de Recherches, Agriculture Canada.     

Native seeds incorporated into activated carbon pods applied concurrently with indaziflam: a new strategy for restoring annual‐invaded communities?

Reestablishing native perennial vegetation in annual grass‐invaded rangelands is critical to restoring ecosystems. Control of exotics, often achieved with preemergent herbicides, is essential for successful restoration of invaded rangelands. Unfortunately, desirable species cannot be seeded simultaneously with preemergent herbicide application due to nontarget damage. To avoid this, seeding is commonly delayed at least 1 year. Delaying seeding increases the likelihood that annual grasses will begin reestablishing and compete with seeded species. Activated carbon (AC) can provide preemergent herbicide protection for seeded species because it adsorbs and deactivates herbicides. Previous studies suggest that a cylindrical herbicide protection pod (HPP), containing AC and seeds, allows desired species to be seeded simultaneously with the application of the preemergent herbicide imazapic. Unfortunately, imazapic is only effective at controlling annual grasses for 1–2 years. Indaziflam is a new preemergent herbicide which exhibits longer soil activity, with which HPPs may be useful. To assess this possibility, we evaluated seeding two native species (Wyoming big sagebrush [Artemisia tridentata Nutt ssp. wyomingensis] and bluebunch wheatgrass [Pseudoroegneria spicata (Pursh) Á. Löve]), both incorporated into HPPs and as bare seed, at four application rates of indaziflam in a grow room study. HPPs protected seeded species at low, mid, and high rates of indaziflam. The abundance and size of plants was greater in HPPs compared to bare seed treatments. These results suggest that HPPs can be used to seed native grasses and shrubs simultaneously with indaziflam application.     

The influence of serum‐supplemented culture media in a transwell migration assay

Our work cautions against the use of serum‐supplemented culture media in a transwell migration assay when using chemoattractants other than FBS. At 24 h, a 5% foetal bovine serum (FBS) gradient caused BV2 microglia to migrate toward the lower compartment of the transwell apparatus. Interestingly, FBS‐supplemented media in the absence of a gradient also resulted in notable microglia migration. Serum can therefore confound the interpretation of a transwell migration assay when another chemoattractant is used.     

Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) - also known as the 'cocktail' approach - is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC-MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of 'cocktail' incubates to analyze the probe metabolites 1'-hydroxymidazolam (CYP3A4), 4'-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1'-hydroxytacrine (CYP1A2) and 4'-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.1mg/mL). The analytical methods employ the same mobile phase, acetonitrile and 0.1% formic acid, under similar LC-MS/MS conditions. In the HT method, the chromatographic method consists of a short robust step-gradient where the probe metabolites are simultaneously and quickly eluted to enhance throughput. The probe metabolites are chromatographically resolved in the LD stage by utilizing a true linear gradient to obtain optimal peak separation. The IC50 data generated by both analytical methods using single incubations versus cocktail incubations for various test compounds are in good agreement (correlation coefficient (r2)>or=0.98). The scientist conducting the analysis is provided with a choice of method selection depending on the stage of the test compound and on whether throughput or minimizing interference from other co-eluting metabolites is the most important criterion.     

Genomic analyses and the origin of the eukaryotes

The availability of whole-genome data has created the extraordinary opportunity to reconstruct in fine details the 'tree of life'. The application of such comprehensive effort promises to unravel the enigmatic evolutionary relationships between prokaryotes and eukaryotes. Traditionally, biologists have represented the evolutionary relationships of all organisms by a bifurcating phylogenetic tree. But recent analyses of completely sequenced genomes using conditioned reconstruction (CR), a newly developed gene-content algorithm, suggest that a cycle graph or 'ring' rather than a 'tree' is a better representation of the evolutionary relationships between prokaryotes and eukaryotes. CR is the first phylogenetic-reconstruction method to provide precise evidence about the origin of the eukaryotes. This review summarizes how the CR analyses of complete genomes provide evidence for a fusion origin of the eukaryotes.     

Furcation, field-splitting, and the evolutionary origins of novelty in arthropod photoreceptors

Arthropod photoreceptor evolution is a prime example of how evolution has used existing components in the origin of new structures. Here, we outline a comparative approach to understanding the mutational origins of novel structures, describing multiple examples from arthropod photoreceptor evolution. We suggest that developmental mechanisms have often split photoreceptors during evolution (field-splitting) and we introduce “co-duplication” as a null model for the mutational origins of photoreceptor components. Under co-duplication, gene duplication events coincide with the origin of a higher level structure like an eye. If co-duplication is rejected for a component, that component probably came to be used in a new photoreceptor through regulatory mutations. If not rejected, a gene duplication mutation may have allowed the component to be used in a new structure. In multiple case studies in arthropod photoreceptor evolution, we consistently reject the null hypothesis of co-duplication of genetic components and photoreceptors. Nevertheless, gene duplication events have in some cases occurred later, allowing divergence of photoreceptors. These studies provide a new perspective on the evolution of arthropod photoreceptors and provide a comparative approach that generalizes to the study of any evolutionary novelty.     

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.Equine grass sickness (EGS, or equine dysautonomia) is a predominantly fatal, rapid multiple system neuropathy of grazing horses with reported incidence rates of 2.1–2.3% (reviewed by (1, 2)). An apparently identical disease occurs in cats, dogs, hares, rabbits, llamas, and possibly sheep (3–9). EGS is associated with chromatolysis of sympathetic and parasympathetic postsynaptic neurons, particularly in the enteric nervous system, as well as autonomic presynaptic and somatic lower motor neurons in the brainstem and spinal cord (10). EGS is subdivided into acute, subacute, and chronic forms according to the severity of clinical signs that largely reflect enteric and autonomic neurodegeneration, including dysphagia, generalized ileus, sweating, salivation, ptosis, rhinitis sicca, and tachycardia. While the etiology of EGS remains unknown, some evidence supports it being a toxic infection with Clostridium botulinum type C or D (11, 12). Ultrastructural studies suggest that the lesion in EGS primarily involves the glycoprotein biosynthetic pathway of specific neurons since the rough endoplasmic reticulum and Golgi complexes are consistently affected, while other organelles, including mitochondria, appear relatively normal (13). However, while the ultrastructural and cellular appearance of affected neurons has been studied extensively, little is known about the molecular mechanisms that contribute to neurodegeneration.The overarching aim of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS using a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. The cranial (superior) cervical ganglion (CCG), which supplies sympathetic innervation to the head and neck, was selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS (Fig. 1 and Supplemental Fig. 1 (14)). Here, proteomic analysis was carried out using isobaric tag for relative and absolute quantitation (iTRAQ) tools, which are now well established in small animal models of human neurodegenerative conditions but which are not routinely utilized in large animal models or large animal intrinsic conditions. This proteomic analysis was coupled with quantitative fluorescent Western blotting (QFWB), immunohistochemistry (IHC), and in silico based techniques in an attempt to identify the molecular pathways and processes that may be contributing to neurodegeneration in EGS. Here, we report widespread changes in the CCG of EGS horses, including significant disruption to a broad range of functional pathways clustering around candidates commonly associated with protein misfolding/aggregation responses in human neurodegenerative conditions.Open in a separate windowFig. 1.Equine grass sickness is a predominantly fatal, acute multiple system neuropathy of grazing horses. (A) Example photograph of a horse exhibiting typical appearance associated with chronic EGS. There is ptosis, and generalized muscle weakness as evidenced by the base narrow stance, low head and neck carriage, and leaning against a wall for support. Generalized muscle atrophy and reduced abdominal volume are also evident. (B) Example BIII-tubulin-stained sections from cranial cervical ganglia (CCG), which are known to exhibit neuronal perturbations in this disease. The visible puncta are BIII positive neurons. (C) High power micrographs stained CCG sections from B showing BIII positive neuronal profiles. (D) Quantification of BIII positive neurons demonstrates that there is still equivalent neuronal density in ganglia at terminal stages of the disease (control 6.25 ± 0.12, EGS 6.10 ± 0.20 cells per 100 μm², mean ± Standard Error (S.E) n = 4 cases per condition, n = 116 grids measured. See Materials and Methods for more information). (E) Example H and E stained sections from control and EGS-affected CCG demonstrates that while the neuronal density may be similar, many of the neurons exhibit chromatolysis. Scale bar = 0.75ft (A), 0.5 cm (B), 35 μm (C), 100 μm (E).This study therefore represents the first application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). It is also the first to demonstrate correlation and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal vulnerability and a range of human neurological conditions from childhood neurodegenerative conditions such as spinal muscular atrophy through to diseases associated with advancing age such as Alzheimer''s. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals.     

Altered Ig hypermutation pattern and frequency in complementary mouse models of DNA polymerase ζ activity

To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.