RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.