Project description:Legionnaire’s Disease is a growing concern for the United States and Europe, with disease incidences rising 6-fold since 2002. These recorded cases are increasingly associated with antibiotic resistant Legionella pneumophila, the causative agent of Legionnaire’s Disease and overall Legionellosis. With this, the need to study L. pneumophilainfections has never been greater. Current methodology for Legionella pneumophila infection studies often revolves around either artificial administration using intranasal or intratracheal delivery, semi-authentic delivery using bioaerosols and individual delivery systems (i.e. nose cones), or the burgeoning field of authentic exposure scenarios using aerosol generating showerhead devices. Here, we developed an alternative method using a Madison Aerosol Chamber as a means of generating and delivering bioaerosols in mice. We show that bioaerosol delivery using the Aerosol Exposure Chamber is very effective at exposing mice to various doses of L. pneumophila. RNASeq analyses revealed a robust immune response to bioaerosol delivered L. pneumophila comprising of activations of classical markers of infection and inflammation, including Cxcl and Ccl family genes and Il-1β. Similar gene expression profiles were observed when animals were intranasally exposed to L. pneumophila. Intranasal delivery resulted in a shorter duration of activation of several genes, indicating a lack of realistic infection response. Taken together, this evidence shows that our system delivers similar, if not better, results than intranasal inoculation while allowing researchers to study bioaerosol generation and delivery mechanisms simultaneously, critical factors for studying Legionella pneumophila infection. Such a new approach will allow for more accurate investigations to understand the effects of inhaling to Legionella contaminated drinking water.
Project description:Metaproteomic analysis of air particulate matter provides information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters was developed. Different protein extraction procedures were tested in order to select the best extraction protocol in terms of protein recovery. The optimized method was tested for extraction of proteins from spores of ubiquitous bacteria species and used for the first time for the metaproteomics characterization of filters from work environment. In particular, ambient aerosol samples were collected in different working environments, i.e. a composting plant, wastewater treatment plant and agricultural holding. One-hundred seventy-nine, 15, 205 and 444 proteins were successfully identified in composting plant, wastewater treatment plant, and agricultural holding, respectively. All identified proteins were mainly originate from fungi, bacteria and plants which is in line with the major categories of primary biological aerosol particles. The paper is the first metaproteomic study applied to bioaereosol samples collected in occupationally relevant environmental sites providing interesting information on the composting, wastewater treatment and feed blending processes. Significance This manuscript describes the metaproteomic analysis of aerosol samples collected in work enviroments. This is a novel use of aereosol samples and is needed as there is no really comprehensive way of analysing aereosol samples from a metaproteomic point of view. This paper could help to advance methods for metaproteomic analysis of bioaersols, specifically by comparing protein extraction protocols and pairing the best performing extraction protocol with a gel-free protein separation procedure applied for the first time for analysis of bioaerosol samples. The obtained data showed as bioaerosol was essentially made of fungi, bacteria and plant proteins, many of which could be associated to possible aerosolisation and could be a major health concern for workers on site and to the populations residing in neighbouring area.
Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
Project description:A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed