Expression of the human β amyloid peptide (Aβ) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular Aβ in vivo . Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with Aβ has identified six likely chaperone proteins: two members of the HSP70 family, three αB-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription–PCR analysis shows that the small heat shock proteins are also transcriptionally induced by Aβ expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular Aβ in this model. Transgenic animals expressing a nonaggregating Aβ variant, a single-chain Aβ dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with Aβ expression. These results suggest that chaperone function can play a role in modulating intracellular Aβ metabolism and toxicity.

Background: Hookworms, infecting over one billion people, are the mostly closely related major human parasites to the model nematode Caenorhabditis elegans. Applying genomics techniques to these species, we analyzed 3,840 and 3,149 genes from Ancylostoma caninum and A. ceylanicum.

Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the Ancylostoma ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-D-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology.

We report on the experimental implementation of the transgenic phiC31 recombinase C. elegans intron-split system. The three-component plasmid-based phiC31 recombinase system consists of i) two intron-split segments of the C. briggsae-unc-119 gene, and ii) the plasmid that provides phiC31 recombinase activity. Described results constitute the proof-of-concept assay for the implementation of bacteriophage phiC31 integrase in C. elegans.

No abstract

RNA-interference (Fire et al. 1998) is a popular ‘reverse-genetics’ screening strategy applied in Caenorhabditis elegans. Genome-wide RNAi screens are presently carried using RNAi feeding libraries. Here, we report on a complementary resource facilitating an approach to RNAi screen relying on an unbiased ‘forward-genetics’ strategy. We conclude the forward RNA interference screening is useful and feasible, with the strong expectation the presented screening mode will complement and extend on the existing, currently available, genome-wide RNAi resources.

Abbreviations: HD -Huntington disease; AD -Alzheimer disease; HTT -huntingtin; APPamyloid precursor protein; Aß42 -ß-amyloid peptide; ER -endoplasmic reticulum; HSP -heat shock protein; YFP -yellow fluorescent protein; co-IP -co-immunoprecipitation; RNAi -RNA interference; polyQ -polyglutamine ABSTRACT Formation of proteinaceous deposits composed of abnormally aggregated proteins characterizes a range of pathological conditions. Proteinaceous inclusions detected in the neurodegenerative conditions such Huntington's chorea (HD) and Alzheimer's disease (AD) are often referred as pathognomic and regarded as causally implicated. Despite of differences in aetiology and underlying genetics, rare cases of combined HD and AD were reported and described as admixed proteinopathies. Mixed proteopathies are characterized by the co-occurrence of at least two types of abnormal aggregation-prone variants; pathological deposition of proteinaceous inclusions might however, affect different cell populations. Here, combining plaques derived from human ß-amyloid with mutant HTT-like polyglutamine inclusions in a cell-autonomous manner, we report on the Caenorhabditis elegans model for admixed proteinopathy of Alzheimer's and Huntington's type. We show both types of intracellular foci are formed in vivo: non-amyloidic extended polyglutamine derived inclusions and distinguish those from the presence of mature ßamyloid fibers. We found that polyglutamines expanded above pathogenic threshold and ß-amyloid act synergistically to promote the progression of proteotoxicity in a temperature dependent manner.We further, implicate the hsp-1 (the predominant C. elegans chaperone interacting with ER-routed Aß42) modulate the proteotoxic insults observed in combined proteopathy model. Our results demonstrate how the in vivo model of admixed proteopathy could be utilized to probe for human pathogenic variants confined to the same cellular type. In that perspective expanded aggregationprone polyglutamines appended with fluorescent proteins could be regarded as 'pathogenic probes' useful in the proteotoxicity assays i.e. involving ß-amyloid and possibly other comparable models of disease-associated aggregation prone variants.We anticipate models of combined proteopathies will be informative regarding the underlying pathogenesis and provide the sensitized background

Our perspective article covers major findings concerning Feline Infectious Peritonitis (FIP) - a fatalcoronaviral disease of cats. In the context of FIP pathogenesis, we outline disease signalment andfocus on the challenges and promises of FIP invoking coronavirus RNA detection. In particular, weoutline critical aspects of coronavirus RNA replication and biogenesis. We infer the replicativeintermediates of feline coronavirus may constitute an underappreciated factor triggering theprogression of the maladaptive immune response underlying FIP pathogenesis.