Inhibition of calcium-dependent protein kinase 1 (CDPK1) in vitro by pyrazolopyrimidine derivatives does not correlate with sensitivity of Cryptosporidium parvum growth in cell culture

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration

Dynamic Changes in the Global MicroRNAome and Transcriptome Identify Key Nodes Associated With Ovarian Development in Chickens

The analysis of gene expression patterns during ovarian follicle development will advance our understanding of avian reproductive physiology and make it possible to improve laying performance. To gain insight into the molecular regulation of ovarian development, a systematic profiling of miRNAs and mRNAs at four key stages was conducted, using ovarian tissues from hens at 60 days of age (A), 100 days (B), 140 days-not yet laying (C), and 140 days-laying (D). Comparisons of consecutive stages yielded 73 differentially expressed miRNAs (DEMs) (14 for B vs. A, 8 for C vs. B, and 51 for D vs. C) and 2596 differentially expressed genes (DEGs) (51 for B vs. A, 20 for C vs. B, and 2579 for D vs. C). In addition, 174 DEMs (22 for C vs. A, 74 for D vs. A, and 78 for D vs. B) and 3205 DEGs (118 for C vs. A, 2284 for D vs. A, and 2882 for D vs. B) were identified between nonconsecutive stages. Some DEGs are involved in the Wnt and TGF-beta signaling pathways, which are known to affect ovarian development and ovulation. An integrative analysis of the miRNA and mRNA profiles identified 3166 putative miRNA-mRNA regulatory pairs containing 84 DEMs and 1047 DEGs. Functional annotation of the networks provides strong evidence that the miRNA regulatory networks may play vital roles in ovarian development and ovulation. Ten DEMs and 10 genes were validated by real-time quantitative PCR. The candidate miRNA-mRNA pairs gga-miR-200a-3p-SFRP4, gga-miR-101-3p-BMP5, gga-miR-32-5p-FZD4, and gga-miR-458b-5p-CTNNB1 potentially associated with ovarian development

Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii

Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at ∼300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of ∼592 cM and an average map unit of ∼104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies

Identification of Small Molecule Inhibitors That Block the Toxoplasma gondii Rhoptry Kinase ROP18

The protozoan parasite Toxoplasma gondii secretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component of T. gondii strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 in vitro and subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon gamma-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δ rop18 mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence

Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility

Gene discovery in the Apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res

Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%-20% represent putative homologs with a conservative cutoff of p < 10 −9 , thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets

Dormancy-Breaking and Germination Requirements for Seeds of <i>Sorbus alnifolia</i> (Siebold &amp; Zucc.) K.Koch (Rosaceae), a Mesic Forest Tree with High Ornamental Potential

Sorbus alnifolia (Siebold &amp; Zucc.) K.Koch (Rosaceae) is an economically important tree in the temperate forests of Eastern China. In recent decades, ever-increasing use and modification of forestlands have resulted in major degeneration of the natural habitat of S. alnifolia. Moreover, S. alnifolia seeds germinate in a complicated way, leading to a high cost of propagation. The current study aimed to determine the requirements for breaking seed dormancy and for germination as well as to characterize the type of seed dormancy present in this species. Moreover, the roles of temperature, cold/warm stratification, and gibberellic acid (GA3) in breaking dormancy were tested combined with a study of the soil seed bank. The results showed that intact seeds of S. alnifolia were dormant, requiring 150 days of cold stratification to achieve the maximum germination percentage at 5/15 &#176;C. Exposure of the seeds to ranges of temperatures at 15/25 &#176;C and 20/30 &#176;C resulted in secondary dormancy. Scarifying seed coat and partial removal of the cotyledon promoted germination. Compared with long-term cold stratification, one month of warm stratification plus cold stratification was superior in breaking dormancy. Application of GA3 did not break the dormancy during two months of incubation. Seeds of S. alnifolia formed a transient seed bank. The viability of freshly matured S. alnifolia seeds was 87.65% &#177; 11.67%, but this declined to 38.25% after 6-months of storage at room temperature. Seeds of S. alnifolia have a deep physiological dormancy; cold stratification will be useful in propagating this species. The long chilling requirements of S. alnifolia seeds would avoid seedling death in winter

Sensitive and Specific Identification of Neospora caninum Infection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle

The neuropsychological profiles and semantic-critical regions of right semantic dementia

Introduction: Previous literature has revealed that the anterior temporal lobe (ATL) is the semantic hub of left-sided or mixed semantic dementia (SD), whilst the semantic hub of right-sided SD has not been examined. Methods: Seventeen patients with right-sided SD, 18 patients with left-sided SD and 20 normal controls (NC) underwent neuropsychological assessments and magnetic resonance imaging scans. We investigated the relationship between the degree of cerebral atrophy in the whole brain and the severity of semantic deficits in left and right-sided SD samples, respectively. Results: We found the semantic deficits of right-sided SD patients were related to bilateral fusiform gyri and left temporal pole, whilst the left fusiform gyrus correlated with the semantic performance of left-sided SD patients. Moreover, all the findings couldn't be accounted for by total gray matter volume (GMV) or general cognitive degradation of patients. Discussion: These results provide novel evidence for the current semantic theory, that the important regions for semantic processing include both anterior and posterior temporal lobes. Keywords: Semantic dementia, Lesion-behavior mapping, Laterality of brain atrophy, Semantic deficit