Cosmological Relativity: A General-Relativistic Theory for the Accelerating Expanding Universe

Recent observations of distant supernovae imply, in defiance of expectations, that the universe growth is accelerating, contrary to what has always been assumed that the expansion is slowing down due to gravity. In this paper a general-relativistic cosmological theory that gives a direct relationship between distances and redshifts in an expanding universe is presented. The theory is actually a generalization of Hubble's law taking gravity into account by means of Einstein's theory of general relativity. The theory predicts that the universe can have three phases of expansion, decelerating, constant and accelerating, but it is shown that at present the first two cases are excluded, although in the past it had experienced them. Our theory shows that the universe now is definitely in the stage of accelerating expansion, confirming the recent experimental results

Conformal Quivers and Melting Molecules

Quiver quantum mechanics describes the low energy dynamics of a system of wrapped D-branes. It captures several aspects of single and multicentered BPS black hole geometries in four-dimensional $\mathcal{N} = 2$ supergravity such as the presence of bound states and an exponential growth of microstates. The Coulomb branch of an Abelian three node quiver is obtained by integrating out the massive strings connecting the D-particles. It allows for a scaling regime corresponding to a deep AdS$_2$ throat on the gravity side. In this scaling regime, the Coulomb branch is shown to be an $SL(2,\mathbb{R})$ invariant multi-particle superconformal quantum mechanics. Finally, we integrate out the strings at finite temperature---rather than in their ground state---and show how the Coulomb branch `melts' into the Higgs branch at high enough temperatures. For scaling solutions the melting occurs for arbitrarily small temperatures, whereas bound states can be metastable and thus long lived. Throughout the paper, we discuss how far the analogy between the quiver model and the gravity picture, particularly within the AdS$_2$ throat, can be taken.Comment: 49 pages, 16 figure

Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies

Stable S/MAR-based episomal vectors are regulated at the chromatin level

Episomal vectors assembled from defined genetic components are a promising alternative to traditional gene therapy vectors that integrate in the host genome and may cause insertional mutations. The vector pEPI-eGFP is stably retained in the episomal state in cultured mammalian cells at low copy number for many generations without integration into the host genome. Although pEPI-eGFP is a fully engineered vector, little is known about how it interacts with the host genome and about the molecular mechanisms that are responsible for its transcriptional activity. We have analyzed the expression of the episomal reporter gene eGFP under conditions that affect the chromatin state of the genome. We have also constructed pEPI derivatives carrying a tandem array of lac operator sequences, which allows in vivo visualization and manipulation of the chromatin state of the episome. We show that changes in chromatin state of both the host and pEPI-eGFP induces changes in episomal gene activity and influences the episome’s nuclear distributions. We conclude that episomal genes are subject to control systems of the host, similarly to their counterparts in the host genome

Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5′-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes

MicroRNA-Restricted Transgene Expression in the Retina

Background: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 39UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additiona

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed

Different reprogramming propensities in plants and mammals: Are small variations in the core network wirings responsible?

Although the plant and animal kingdoms were separated more than 1,6 billion years ago, multicellular development is for both guided by similar transcriptional, epigenetic and posttranscriptional machinery. One may ask to what extent there are similarities and differences in the gene regulation circuits and their dynamics when it comes to important processes like stem cell regulation. The key players in mouse embryonic stem cells governing pluripotency versus differentiation are Oct4, Sox2 and Nanog. Correspondingly, the WUSCHEL and CLAVATA3 genes represent a core in the Shoot Apical Meristem regulation for plants. In addition, both systems have designated genes that turn on differentiation. There is very little molecular homology between mammals and plants for these core regulators. Here, we focus on functional homologies by performing a comparison between the circuitry connecting these players in plants and animals and find striking similarities, suggesting that comparable regulatory logics have been evolved for stem cell regulation in both kingdoms. From in silico simulations we find similar differentiation dynamics. Further when in the differentiated state, the cells are capable of regaining the stem cell state. We find that the propensity for this is higher for plants as compared to mammalians. Our investigation suggests that, despite similarity in core regulatory networks, the dynamics of these can contribute to plant cells being more plastic than mammalian cells, i.e. capable to reorganize from single differentiated cells to whole plants-reprogramming. The presence of an incoherent feed-forward loop in the mammalian core circuitry could be the origin of the different reprogramming behaviour.This work was supported Swedish Research Council, grant VR 621-2013-4547 to CP; the Swedish Foundation for Strategic Research, grant A3 04 159p to CP; the Gatsby Charitable Foundation (GB), grant GAT3395-PR4 to HJ and the Swedish Research Council, grant VR 621- 2013-4632 to HJ