Ups and downs of RNA interference in parasitic nematodes.

RNA interference (RNAi) is widely used in Caenorhabiditis elegans to identify essential gene function. In parasitic nematodes RNAi has been reported to result in transcript knockdown of some target genes, but not others, thus limiting its use as a potential functional genomics tool. We recently extended work in Haemonchus contortus to examine why only some genes seem to be susceptible to RNAi and to test RNAi effects in vivo. Here we review our findings, which suggest that site of gene expression influences silencing. This most likely reflects limited uptake of dsRNA from the environment, a phenomenon also observed in other free-living nematodes. We discuss new technologies to improve dsRNA delivery, such as nanoparticles being developed for therapeutic siRNA delivery, and methods to monitor RNAi effects. Alternative approaches will be important in progressing the application of RNAi to identify essential gene function in parasitic nematodes.

Toxoplasmosis awareness, seroprevalence and risk behavior among pregnant women in the Gampaha district, Sri Lanka.

BACKGROUND: Primary gestational toxoplasmosis can be transmitted to the fetus with deleterious effects on the pregnancy. There is very little information regarding gestational toxoplasmosis in Sri Lanka. This survey was done to determine the prevalence and awareness of toxoplasmosis and to identify risk factors of infection among pregnant women in the Gampaha district, Sri Lanka. METHODS: Women attending obstetric clinics at the Colombo North Teaching Hospital in 2014 were tested for Toxoplasma gondii (T. gondii) specific Immunoglobulins G (IgG) and M (IgM) subtypes using the OnSite Toxo IgG/IgM Rapid Test-Dip Strip(®). Disease awareness and risk behaviors of the participants were investigated. RESULTS: Of the 293 participants (mean age 27 years, SD ± 5.92), 38% were primigravidae with a mean gestational age of 16.2 weeks (SD 7). The prevalence of anti-T. gondii IgG and IgM antibodies was 12.3% (n = 36) and zero, respectively. Unadjusted and adjusted odds ratios were calculated to determine risk factors of infection (cat-ownership, handling cats, consumption of meat, commercial meals and unwashed raw vegetables and fruits, handling soil and not washing hands after handling soil). On bivariate analysis, eating commercially prepared meals weekly or more was associated with toxoplasma seroprevalence with marginal statistical significance. On multivariate analysis, none of the considered risk factors were significant. Toxoplasma awareness was 4.4% (n = 13); health personnel (46.2%, n = 6) and media (53.8%, n = 7) being sources of information. CONCLUSIONS: Health education programs to increase awareness of toxoplasmosis is recommended at antenatal clinics.

Heat shock factor 1 confers resistance to Hsp90 inhibitors through p62/SQSTM1 expression and promotion of autophagic flux.

Heat shock protein 90 (Hsp90) has an important role in many cancers. Biochemical inhibitors of Hsp90 are in advanced clinical development for the treatment of solid and hematological malignancies. At the cellular level, their efficacy is diminished by the fact that Hsp90 inhibition causes activation of heat shock factor 1 (HSF1). We report a mechanism by which HSF1 activation diminishes the effect of Hsp90 inhibitors geldanamycin and 17-allylaminogeldanamycin (17-AAG, tanespimycin). Silencing HSF1 with siRNA or inhibiting HSF1 activity with KRIBB11 lowers the threshold for apoptosis in geldanamycin and 17-AAG-treated cancer cells. Autophagy also mitigates the actions of Hsp90 inhibitors. Blocking autophagy with 3-methyladenine (3-MA), bafilomycin A1, or beclin 1 siRNA also lower the threshold for apoptosis. Exploring a potential relationship between HSF1 and autophagy, we monitored autophagosome formation and autophagic flux in control and HSF1-silenced cells. Results show HSF1 is required for autophagy in Hsp90 inhibitor-treated cells. The reduced autophagy observed in HSF1-silenced cells correlates with enhanced cell death. To investigate how HSF1 promotes autophagy, we monitored the expression of genes involved in the autophagic cascade. These data show that sequestosome 1 (p62/SQSTM1), a protein involved in the delivery of autophagic substrates and nucleation of autophagosomes, is an HSF1-regulated gene. Gene silencing was used to evaluate the significance of p62/SQSTM1 in Hsp90 inhibitor resistance. Cells where p62/SQSTM1 was silenced showed a dramatic increase in sensitivity to Hsp90 inhibitors. Results highlight the importance of HSF1 and HSF1-dependent p62/SQSTM1 expression in resistance Hsp90 inhibitors, underscoring the potential of targeting HSF1 to improve the efficacy of Hsp90 inhibitors in cancer.

Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene.

Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of ß-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, ß-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites.