Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 μM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART

Historicising Material Agency: from Relations to Relational Constellations

Relational approaches have gradually been changing the face of archaeology over the last decade: analytically, through formal network analysis; and interpretively, with various frameworks of human-thing relations. Their popularity has been such, however, that it threatens to undermine their relevance. If everyone agrees that we should understand past worlds by tracing relations, then ‘finding relations’ in the past becomes a self-fulfilling prophecy. Focusing primarily on the interpretive approaches of material culture studies, this article proposes to counter the threat of irrelevance by not just tracing human-thing relations, but characterising how sets of relations were ordered. Such ordered sets are termed ‘relational constellations’. The article describes three relational constellations and their consequences based on practices of fine ware production in the Western Roman provinces (first century BC – third century AD): the fluid, the categorical, and the rooted constellation. Specifying relational constellations allows reconnecting material culture to specific historical trajectories, and offers scope for meaningful cross-cultural comparisons. As such a small theoretical addition based on the existing toolbox of practice-based approaches and relational thought can impact on historical narratives, and can save relational frameworks from the danger of triviality.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s10816-015-9244-

The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease.

The miR-17/92 cluster is among the best-studied microRNA clusters. Interest in the cluster and its members has been increasing steadily and the number of publications has grown exponentially since its discovery with more than 1000 articles published in 2012 alone. Originally found to be involved in tumorigenesis, research work in recent years has uncovered unexpected roles for its members in a wide variety of settings that include normal development, immune diseases, cardiovascular diseases, neurodegenerative diseases and aging. In light of its ever-increasing importance and ever-widening regulatory roles, we review here the latest body of knowledge on the cluster\u27s involvement in health and disease as well as provide a novel perspective on the full spectrum of protein-coding and non-coding transcripts that are likely regulated by its members

Circulating microRNAs as novel biomarkers for diabetes mellitus.

Diabetes mellitus is characterized by insulin secretion from pancreatic β cells that is insufficient to maintain blood glucose homeostasis. Autoimmune destruction of β cells results in type 1 diabetes mellitus, whereas conditions that reduce insulin sensitivity and negatively affect β-cell activities result in type 2 diabetes mellitus. Without proper management, patients with diabetes mellitus develop serious complications that reduce their quality of life and life expectancy. Biomarkers for early detection of the disease and identification of individuals at risk of developing complications would greatly improve the care of these patients. Small non-coding RNAs called microRNAs (miRNAs) control gene expression and participate in many physiopathological processes. Hundreds of miRNAs are actively or passively released in the circulation and can be used to evaluate health status and disease progression. Both type 1 diabetes mellitus and type 2 diabetes mellitus are associated with distinct modifications in the profile of miRNAs in the blood, which are sometimes detectable several years before the disease manifests. Moreover, circulating levels of certain miRNAs seem to be predictive of long-term complications. Technical and scientific obstacles still exist that need to be overcome, but circulating miRNAs might soon become part of the diagnostic arsenal to identify individuals at risk of developing diabetes mellitus and its devastating complications

Bistability and Oscillations in Gene Regulation Mediated by Small Noncoding RNAs

The interplay of small noncoding RNAs (sRNAs), mRNAs, and proteins has been shown to play crucial roles in almost all cellular processes. As key post-transcriptional regulators of gene expression, the mechanisms and roles of sRNAs in various cellular processes still need to be fully understood. When participating in cellular processes, sRNAs mainly mediate mRNA degradation or translational repression. Here, we show how the dynamics of two minimal architectures is drastically affected by these two mechanisms. A comparison is also given to reveal the implication of the fundamental differences. This study may help us to analyze complex networks assembled by simple modules more easily. A better knowledge of the sRNA-mediated motifs is also of interest for bio-engineering and artificial control

Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type

Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21–24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches

Sense and Antisense Transcripts of Convergent Gene Pairs in Arabidopsis thaliana Can Share a Common Polyadenylation Region

The Arabidopsis genome contains a large number of gene pairs that encode sense and antisense transcripts with overlapping 3′ regions, indicative for a potential role of natural antisense transcription in regulating sense gene expression or transcript processing. When we mapped poly(A) transcripts of three plant gene pairs with long overlapping antisense transcripts, we identified an unusual transcript composition for two of the three gene pairs. Both genes pairs encoded a class of long sense transcripts and a class of short sense transcripts that terminate within the same polyadenylation region as the antisense transcripts encoded by the opposite strand. We find that the presence of the short sense transcript was not dependent on the expression of an antisense transcript. This argues against the assumption that the common termination region for sense and antisense poly(A) transcripts is the result of antisense-specific regulation. We speculate that for some genes evolution may have especially favoured alternative polyadenylation events that shorten transcript length for gene pairs with overlapping sense/antisense transcription, if this reduces the likelihood for dsRNA formation and transcript degradation

An Inhibitory Effect of Extracellular Ca2+ on Ca2+-Dependent Exocytosis

Aim: Neurotransmitter release is elicited by an elevation of intracellular Ca 2+ concentration ([Ca 2+] i). The action potential triggers Ca 2+ influx through Ca 2+ channels which causes local changes of [Ca 2+] i for vesicle release. However, any direct role of extracellular Ca 2+ (besides Ca 2+ influx) on Ca 2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis. Results: Using photolysis of caged Ca 2+ and caffeine-induced release of stored Ca 2+, we found that extracellular Ca 2+ inhibited exocytosis following moderate [Ca 2+]i rises (2–3 mM). The IC50 for extracellular Ca 2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (,30%) of extracellular Ca 2+ concentration ([Ca 2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca 2+] o. The calcimimetics Mg 2+,Cd 2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca 2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. Conclusion/Significance: As an extension of the classic Ca 2+ hypothesis of synaptic release, physiological levels of extracellular Ca 2+ play dual roles in evoked exocytosis by providing a source of Ca 2+ influx, and by directly regulatin

The Genetic Signatures of Noncoding RNAs

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses