Cross Platform Standardisation of an Experimental Pipeline for Use in the Identification of Dysregulated Human Circulating MiRNAs.

INTRODUCTION: Micro RNAs (miRNAs) are a class of highly conserved small non-coding RNAs that play an important part in the post-transcriptional regulation of gene expression. A substantial number of miRNAs have been proposed as biomarkers for diseases. While reverse transcriptase Real-time PCR (RT-qPCR) is considered the gold standard for the evaluation and validation of miRNA biomarkers, small RNA sequencing is now routinely being adopted for the identification of dysregulated miRNAs. However, in many cases where putative miRNA biomarkers are identified using small RNA sequencing, they are not substantiated when RT-qPCR is used for validation. To date, there is a lack of consensus regarding optimal methodologies for miRNA detection, quantification and standardisation when different platform technologies are used. MATERIALS AND METHODS: In this study we present an experimental pipeline that takes into consideration sample collection, processing, enrichment, and the subsequent comparative analysis of circulating small ribonucleic acids using small RNA sequencing and RT-qPCR. RESULTS, DISCUSSION, CONCLUSIONS: Initially, a panel of miRNAs dysregulated in circulating blood from breast cancer patients compared to healthy women were identified using small RNA sequencing. MiR-320a was identified as the most dysregulated miRNA between the two female cohorts. Total RNA and enriched small RNA populations (<30 bp) isolated from peripheral blood from the same female cohort samples were then tested for using a miR-320a RT-qPCR assay. When total RNA was analysed with this miR-320a RT-qPCR assay, a 2.3-fold decrease in expression levels was observed between blood samples from healthy controls and breast cancer patients. However, upon enrichment for the small RNA population and subsequent analysis of miR-320a using RT-qPCR, its dysregulation in breast cancer patients was more pronounced with an 8.89-fold decrease in miR-320a expression. We propose that the experimental pipeline outlined could serve as a robust approach for the identification and validation of small RNA biomarkers for disease.

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water.

BACKGROUND: Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW. RESULTS: A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively. Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines. CONCLUSIONS: The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.

Overview of invasive fungal infections.

The incidence of invasive fungal infections (IFIs) has seen a marked increase in the last two decades. This is especially evident among transplant recipients, patients suffering from AIDS, in addition to those in receipt of immunosuppressive therapy. Worryingly, this increased incidence includes infections caused by opportunistic fungi and emerging fungal infections which are resistant to or certainly less susceptible than others to standard antifungal agents. As a direct response to this phenomenon, there has been a resolute effort over the past several decades to improve early and accurate diagnosis and provide reliable screening protocols thereby promoting the administration of appropriate antifungal therapy for fungal infections. Early diagnosis and treatment with antifungal therapy are vital if a patient is to survive an IFI. Substantial advancements have been made with regard to both the diagnosis and subsequent treatment of an IFI. In parallel, stark changes in the epidemiological profile of these IFIs have similarly occurred, often in direct response the type of antifungal agent being administered. The effects of an IFI can be far reaching, ranging from increased morbidity and mortality to increased length hospital stays and economic burden.

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA.

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis.

In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, alpha-keto-beta-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.