Mammalian cells in culture actively export specific microRNAs

The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research, but has left many unanswered questions about how this regulation works and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in blood plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations; and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication^[1-5]^. Such a system would be expected to export specific miRNAs from cells in response to specific biological stimuli. We report here that after serum deprivation several human cell lines tested do export a spectrum of miRNAs into the culture medium. The export response is substantial and prompt. The exported miRNAs are found both within and outside of microvesicles and exosomes. We have identified some candidate protein components of this system outside the cell, and found one exported protein that plays a role in protecting miRNA from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that may involve cell-cell communication

Describing the complexity of systems: multi-variable "set complexity" and the information basis of systems biology

Context dependence is central to the description of complexity. Keying on the pairwise definition of "set complexity" we use an information theory approach to formulate general measures of systems complexity. We examine the properties of multi-variable dependency starting with the concept of interaction information. We then present a new measure for unbiased detection of multi-variable dependency, "differential interaction information." This quantity for two variables reduces to the pairwise "set complexity" previously proposed as a context-dependent measure of information in biological systems. We generalize it here to an arbitrary number of variables. Critical limiting properties of the "differential interaction information" are key to the generalization. This measure extends previous ideas about biological information and provides a more sophisticated basis for study of complexity. The properties of "differential interaction information" also suggest new approaches to data analysis. Given a data set of system measurements differential interaction information can provide a measure of collective dependence, which can be represented in hypergraphs describing complex system interaction patterns. We investigate this kind of analysis using simulated data sets. The conjoining of a generalized set complexity measure, multi-variable dependency analysis, and hypergraphs is our central result. While our focus is on complex biological systems, our results are applicable to any complex system.Comment: 44 pages, 12 figures; made revisions after peer revie

Comparison of reproducibility, accuracy, sensitivity, and specificity of miRNA quantification platforms

Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use

Small RNA profiling of low biomass samples: identification and removal of contaminants

Background Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation. Results Herein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using ‘ultra-clean’ extraction kits. Conclusion This is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies. Keywords: RNA sequencing; Artefact removal; Exogenous RNA in human blood plasma; Contaminant RNA; Spin column

SRM targeted proteomics in search for biomarkers of HCV ‐induced progression of fibrosis to cirrhosis in HALT ‐ C patients

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92042/1/pmic7038.pd

Atypical, non-standard functions of the microtubule associated Tau protein.

Since the discovery of the microtubule-associated protein Tau (MAPT) over 40 years ago, most studies have focused on Tau's role in microtubule stability and regulation, as well as on the neuropathological consequences of Tau hyperphosphorylation and aggregation in Alzheimer's disease (AD) brains. In recent years, however, research efforts identified new interaction partners and different sub-cellular localizations for Tau suggesting additional roles beyond its standard function as microtubule regulating protein. Moreover, despite the increasing research focus on AD over the last decades, Tau was only recently considered as a promising therapeutic target for the treatment and prevention of AD as well as for neurological pathologies beyond AD e.g. epilepsy, excitotoxicity, and environmental stress. This review will focus on atypical, non-standard roles of Tau on neuronal function and dysfunction in AD and other neurological pathologies providing novel insights about neuroplastic and neuropathological implications of Tau in both the central and the peripheral nervous system