Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

MicroRNAs (miRNAs) are small (～22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.     

Multi-element fingerprinting and high throughput sequencing identify multiple elements that affect fungal communities in Quercus macrocarpa foliage

Diverse fungal mutualists, pathogens and saprobes colonize plant leaves. These fungi face a complex environment, in which stochastic dispersal interplays with abiotic and biotic filters. However, identification of the specific factors that drive the community assembly seems unattainable. We mined two broad data sets and identified chemical elements, to which dominant molecular operational taxonomic units (OTUs) in the foliage of a native tree respond most extremely. While many associations could be identified, potential complicating issues emerged. Those were related to unevenly distributed OTU frequency data, a large number of potentially explanatory variables and the disproportionate effects of outlier observations.Key words: community assembly, environmental filter, fungi, heavy metal enrichment, nutrient enrichment, oak, Quercus macrocarpaHyperdiverse fungal communities inhabit the foliage of most plants^1,2 and these fungal communities have been reported for virtually every plant that has been examined.³ Baas-Becking hypothesis states that environment selects microbial communities from the abundant and possibly globally distributed propagule pools.⁴ Although the foliage-associated communities—like other microbial communities—are suspected to be sensitive to environmental drivers, determination of the mechanisms that control the assembly of these foliar communities has remained difficult and elusive. Some of the proposed mechanisms include distance limitations to propagule dispersal,^5–7 volume limitations to propagule loads,⁷ or limitations set by the environmental conditions either on the scale of the site of fungal colonization⁸ or more broadly on a landscape level.^6,9 The forces that may control the fungal community assembly are overlaid by additional biotic controls that include compatibilities between the fungi and host species^10,11 or genotypes^6,12 and the competitive or facilitative interactions among the component fungal genotypes.^6,10–13 Although a variety of potential controls for the foliage-associated fungal communities have been speculated, very little consensus exists on the relative importance of the different drivers. For example, while macronutrient and heavy metal enrichment may have an influence on the composition fungal communities¹⁴ and populations,¹⁵ relative importance of various chemical elements in the foliage remains yet to be investigated.To evaluate the use of multi-element fingerprinting data produced by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in combination with high throughput 454-pyrosequencing for determining influential chemical elements in structuring of the leaf-associated fungal communities, we mined a recent dataset¹⁶ that explored the effects of urbanization on the diversity and composition of the fungal communities associated with a native tree Quercus macrocarpa. From a total list of more than 700 non-singleton fungal OTUs, we selected fifty with highest overall frequency to provide an observationrich dataset for elemental effect assessment; these OTUs accounted for 84.5% of all sequences. Even so, many of these OTUs had a number of zero frequencies (Fig. 1), highlighting one of the difficulties in the use of environmental sequencing data. We omitted one OTU (OTU630 with a likely affinity to Trimmatostroma cordae [Mycosphaerellaceae]) that was strongly affected by the original land use design (urbanization; Wilcoxon rank sum test with a Bonferroni adjustment) and therefore unlikely to be representative for the present analyses of elemental drivers. This OTU was replaced with one with the next highest frequency. The frequencies of these 50 OTUs were investigated in the context of concentrations for 29 elements after the omission of five (Ag, Au, C, δ¹³C, δ¹⁵N) in the final analyses because of their strong association with the land use or the difficulty of finding a biological relevance. Of the remaining elements three (Fe, Cr and Ni) had pairwise correlations exceeding 0.98 between the three pairings; others showed no similar high correlations. To allow comparable evaluation across the broad array of elements, all concentrations were standardized to have a mean equal to zero and a standard deviation equal to one.Open in a separate windowFigure 1Rank-ordered distribution of observed frequencies for those OTU s whose frequency had an extreme slope when associated with the concentrations of one or more chemical elements in the mixed effects model. The asterisk denotes one extreme frequency for OTU 313 with a value 0.8636. Numbers in parentheses indicate the number of observations with a frequency equal to zero. The OTU s were assigned to approximate taxa using BLAST:²⁰ 425: Alternaria alternata (Pleosporaceae); 46: Phoma glomerata (Pleosporaceae); 686: Aureobasidium pullulans (Dothioraceae); 520: Davidiella tassiana (Davidiellaceae); 567: Cladosporioum tenuissimum (Davidiellaceae); 313 Oidium heveae (mitosporic Erysiphaceae); 586: Erysiphe hypogena (Erysiphaceae); 671: Mycosphaerella microsora (Mycosphaerellaceae); 555: Pestalotiopsis sp. (Amphisphaeriaceae); 607: Pleiochaeta setosa (incertae sedis).To rank elements according to their magnitude of association with the abundance of each OTU, a total of 1,450 models (50 OTUs times 29 elements) relating element concentration to OTU abundance were fit to the data. For each model, OTU frequency was the dependent variable, element concentration and time (a factor with three levels) were fixed effects, and—to account for the spatial arrangement of the experimental units—random effects associated with tree nested within site were included in the error structure. Time by element interactions were also investigated and tested using a likelihood ratio test. These mixed effect models were fit using R and the package lme4 (www.rproject.org).Statistical “tests of significance” that produce p-values can be sensitive to assumptions or outliers. Because of this and the fact that our analyses evaluated a total of 1,450 models, p-values themselves were not considered a reliable measure of importance when associating elements with OTU frequency. Instead, we emphasized metrics that highlight extraordinary findings rather than rely on tests of statistical significance. This approach facilitates finding few elements that have the strongest effect on OTU frequency. Note that the use of standardized element concentrations (above) provided slope coefficients that are comparable across all models. “Extreme slopes”, i.e., models where the OTU response to element concentration was strongest, were identified as those with estimated slope coefficients in the lower or upper 2.5 percentile, i.e., those farther than 1.8 standard deviations from the mean across all estimated slopes (Fig. 2). Using this approach, we identified a total of 69 models with extreme slopes (Open in a separate windowFigure 2Distribution of estimated slopes (i.e., the slope for element concentration) for a model relating OTU frequency to element concentration, time and a concentration by time interaction, including a tree-nested-within-site random effect. The mean across all 1,450 OTU s is approximately zero; the two vertical lines identify upper and lower 2.5 percentiles, beyond which the slopes were considered extreme (large black symbols). The horizontal line identifies the cut off maximum leverage (0.24), above which the slopes were considered to have observations with high leverage. Models with observations with a high leverage were tested for extreme slopes by refitting without those observations. Models are ranked from bottom to top in order of increasing leverage and the element for which the high-leverage observations and extreme slopes were recorded are identified on the right y-axis.

Table 1

Slopes identified as extreme in our analyses

HIV capsid is a tractable target for small molecule therapeutic intervention

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.     

RGS9-2 is a negative modulator of mu-opioid receptor function

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.     

Virus-specific CD8+ T cells in the liver: armed and ready to kill

Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2D(b)NP(366-374) (D(b)NP(366)) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-gamma secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a "graveyard" for influenza virus-specific CD8+ T cells.     

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Delta mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Delta cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.     

Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.     

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.