Daniel P Tonge

Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific “conserved” primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.

β2-Adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle does not modulate disease severity in the rodent meniscectomy model of osteoarthritis

Summary Objective To examine whether β2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model. Methods Male Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n = 15) or saline vehicle (n = 20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n = 10; saline pre-treated n = 10) or a sham control surgical procedure (saline pre-treated n = 5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint. Results Clenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery. Conclusions Our data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to “slow twitch” in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability.

The role of microRNAs in the pathogenesis of MMPi-induced skin fibrodysplasia

BackgroundMatrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in extracellular matrix (ECM) homeostasis. MMPs have been an attractive pharmacological target for a number of indications. However, development has been hampered by the propensity of compounds targeting these enzymes to cause connective-tissue pathologies. The broad-spectrum MMP-inhibitor (MMPi) AZM551248 has been shown to induce such effects in the dog. Histopathological changes were consistent with fibrodysplasia (FD), characterised by fibroblast proliferation and the deposition of collagen in the subcutaneous tissues. We conducted a time-course study administering 20mg/kg/day AZM551248 between 4 and 17 days. Cervical subcutaneous tissue and plasma were sampled during the time-course. miRNA expression profiles in subcutaneous skin specimens following the administration of AZM551248 were determined by high-throughput-sequencing.ResultsAn increasing number of miRNAs were differentially expressed compared with vehicle treated control animals as the study progressed. Several of these were members of the miR-200 family and were significantly attenuated in response to MMPi. As the severity of FD increased at the later time-points, other miRNAs associated with TGFβ synthesis and regulation of the acute inflammatory response were modulated. Evidence indicative of epithelial to mesenchymal transition was present at all study time points. Receiver operator curve (ROC) analysis revealed that miR-21 expression in the cervical subcutaneous tissue was a sensitive and specific biomarker of FD incidence.ConclusionsOur data reveal significant perturbations in canine skin miRNA expression in response to MMPi administration. Furthermore, we have identified dysregulated miRNAs that are associated with processes relevant to the key histopathological events of MMPi-induced FD.

Multi-Method Characterisation of the Human Circulating Microbiome

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonisation of various body niches (e.g. the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other “classically sterile” locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to provide a comprehensive description of the human blood microbiome using a range of complementary molecular and classical microbiological techniques. DNA-level analyses involved the amplification and sequencing of the 16S rDNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA / RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

Molecular Characterisation of the Synovial Fluid Microbiome

Background The term “microbiome” describes the genetic material encoding the various microbial populations that inhabit our body. In contrast, the term “microbiota” refers to the viable organisms that comprise these communities. Whilst the colonisation of specific body sites in contact with the external environment (such as the gastrointestinal tract, skin and vagina) by microorganisms is both well-described and universally accepted, the existence of microbial evidence in other “classically sterile” locations including the blood, synovial space, and lungs, is a relatively new concept. Increasingly, a role for the microbiome in disease initiation and progression is being considered, and it is therefore of upmost importance to increase our understanding of these complex communities. To date, no study has investigated the potential for a “synovial fluid microbiome” using the cutting edge molecular and bioinformatic techniques now available. To this end, we investigated the presence of bacterial and fungal DNA in the synovial fluid of rheumatoid arthritis (RA) patients and healthy control subjects. Methods Synovial fluid samples from 25 subjects were analysed through amplification and sequencing of the 16S rRNA V4 and ITS2 genes followed by bioinformatic analysis. Results Bacterial 16S rRNA was detected in fourteen out of sixteen (87.5%) RA patients, and nine out of nine (100%) healthy control subjects. Fungal DNA was identified in 12 out of 16 (75%) RA samples, and 8 out of 9 (88.8%) healthy controls. Bioinformatic analysis revealed evidence of a complex bacterial and fungal microbiome present in the synovial fluid of both healthy control subjects and RA patients. Concluding remarks Through taxonomic classification of sequencing reads we describe a complex community of bacterial and fungal DNA present in the synovial fluid of both healthy control subjects and RA patients. Through statistical analysis of read abundances, we identify key taxa that are differentially present or abundant dependent on disease status. This is the first study to report the synovial fluid microbiome, and to determine that this is modulated by disease status (RA) as are other classical microbiome niches.

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of adults with poorly controlled asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 17A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFa, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, assessment of the circulating “blood microbiome” was performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in the plasma in a condition-specific manner, and many more were found to be expressed at altered levels. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthma state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

Evidence based housekeeping gene selection for microRNA-sequencing (miRNA-seq) studies

The accurate determination of miRNA expression by PCR requires robust data normalisation. The most commonly used approach involves the use of housekeeping or normalising factors which are assumed invariant across the range of experimental conditions investigated. This assumption is often not proven in all experimental situations, therefore we consider whether global miRNA expression data sets obtained by high throughput sequencing can be screened to identify candidate housekeeping miRNAs (HK-miRNA) for further evaluation. To establish this method, we assessed global miRNA sequencing data from a study involving 30 canine skin specimens. From an initial pool of over 200 miRNAs, we identified several candidate HK-miRNA which demonstrated stability across all study samples using both a classical statistical approach and the NormFinder algorithm. We verified these putative HK-miRNAs using real-time PCR assays to further validate their suitability in this specific experimental setup. Our analysis provides a framework to allow researchers to exploit high-throughput sequencing data (be it their own, or from the short read archive or other genomic repository) to guide HK-miRNA selection.