Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow

Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will en-courage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions

A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes

A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1-2, d 3-4, and d 5-7 (early CL, eCL), d 8-12 (mid CL, mCL), d 13-16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, -2898, -96, -7-5p, -183-5p, -182, and -202 showed drastic up-regulation with a fold-change of >= 2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility

Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms

RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine \RNA\ quality are based on electrophoresis and spectrophotometer assessment, namely A260/A280 and A260/A230 ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, \RNA\ extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded \RNA\ extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted \RNA\ yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor \RNA\ extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted \RNA\ from bacterial cultures seem to be insufficient to ensure reliable gene expression determination.This work was funded by the Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309. The following authors had an individual FCT fellowship: VC (SFRH/BD/78235/2011); LDRM (SFRH/BD/66166/2009)

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 µg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood

Figure 7—figure supplement 3. NDI1 expressing A549 cells are refractory to metformin treatment in a xenograft model of tumor growth.

Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I

Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states