Discovery of a novel species,Theileria haneyi n. sp., infective to equids,highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance

A novel apicomplexan parasite was serendipitously discovered in horses at the United States – Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33?million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.     

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity

Background and aims Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin—lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose).Methods Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed.Key Results Cress-seed exudate contained low-M_r, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µm LMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate.Conclusions LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.     

Genetics of metalaxyl resistance in Phytophthora infestans.

Several sexual crosses involving isolates of Phytophthora infestans of diverse sensitivities to metalaxyl were studied. Metalaxyl sensitivity was determined by comparing the growth of an isolate on metalaxyl-amended agar medium (5 microg/ml) with growth on medium containing no metalaxyl. When both parents had the same phenotype for metalaxyl sensitivity (both resistant or both sensitive), all F1 progeny had the parental phenotype. In two crosses (75 and 76) each involving one sensitive and one resistant parent, however, the progeny segregated 1:1, suggesting that the common resistant parent (Bg8) was heterozygous for metalaxyl sensitivity. When an F2 progeny was constructed from resistant F1 isolates in cross 76, the progeny segregated 1:3 (sensitive:resistant), indicating that metalaxyl resistance in Bg8 is conferred by a single dominant gene. Variation in the progeny sensitivity appears to involve minor genes. A correlation study between metalaxyl resistance and fitness components did not reveal any association.     

Characterization of Six Bacteriophages of Serratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10⁻⁹ to 3.9 × 10⁻⁷, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.     

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton’s tyrosine kinase (BTK)

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459 pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26 pmol/mg protein.     

A general method for assaying homo-and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Transglycanases(endotransglycosylases) cleave a polysaccharide(donor-substrate) in mid-chain, and then transfer a portion onto another poly-or oligosaccharide(acceptor-substrate). Such enzymes contribute to plant cellwall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated3 Hpolysaccharides from unreacted3H-oligosaccharides—the former immobilized(on filter-paper, silica-gel or glassfiber),the latter eluted. On filter-paper, certain polysaccharides [e.g.(1!3, 1!4)-b-D-glucans] remained satisfactorily adsorbed when water-washed; others(e.g. pectins) were partially lost. Many oligosaccharides(e.g. arabinan-, galactan-, xyloglucan-based) were successfully eluted in appropriate solvents, but others(e.g. [3H]xylohexaitol, [3H]mannohexaitol[3H]cellohexaitol) remained immobile. On silica-gel, all3 Holigosaccharides left an immobile ‘ghost' spot(contaminating any3H-polysaccharides), which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glassfiber(GF), onto which the reactionmixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris transb-mannanase. In conclusion, our novel GF-blotting technique ef ficiently frees transglycanase-generated3H-polysaccharides from unreacted3H-oligosaccharides,enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donorand acceptor-substrates.