MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

MicroRNA-199a induces differentiation of induced pluripotent stem cells into endothelial cells by targeting sirtuin 1

The ability to reprogram induced pluripotent stem (iPS) cells from somatic cells may facilitate significant advances in regenerative medicine. MicroRNAs (miRNAs) are involved in a number of core biological processes, including cardiogenesis, hematopoietic lineage differentiation and oncogenesis. An improved understanding of the complex molecular signals that are required for the differentiation of iPS cells into endothelial cells (ECs) may allow specific targeting of their activity in order to enhance cell differentiation and promote tissue regeneration. The present study reports that miR‑199a is involved in EC differentiation from iPS cells. Augmented expression of miR‑199a was detected during EC differentiation, and reached higher levels during the later stages of this process. Furthermore, miR‑199a inhibited the differentiation of iPS cells into smooth muscle cells. Notably, sirtuin 1 was identified as a target of miR‑199a . Finally, the ability of miR‑199a to induce angiogenesis was evaluated in vitro, using Matrigel plugs assays. This may indicate a novel function for miR‑199a as a regulator of the phenotypic switch during vascular cell differentiation. The present study provides support to the notion that with an understanding of the molecular mechanisms underlying vascular cell differentiation, stem cell regenerative therapy may ultimately be developed as an effective treatment for cardiovascular disease.

Rescattering-enhanced dissociation of a molecular ion

Dissociation of the CO2+ ion has been investigated in an intense ultrafast (55 fs) laser field by employing an intensity-selective scan technique and comparing the signals from linearly and circularly polarized pulses. Nonsequential contributions have been observed, highlighting the role of rescattering in the dissociative process.

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells.

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.

Evidence for rescattering in molecular dissociation

Dissociation of the singly ionized CO2+ ion has been investigated in an intense ultrafast (55 fs) laser field, by employing an intensity selective scan technique and comparing the signals from linearly and circularly polarized pulses. Non-sequential contributions have been observed unambiguously for the first time, highlighting the role of rescattering in the dissociative process.

Vitamin D Receptor Modulates the Neoplastic Phenotype Through Antagonistic Growth Regulatory Signals

Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1 alpha,25-(OH)(2) D-3 or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1 alpha,25-(OH)(2) D-3 or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation. (C) 2009 Wiley-Liss, Inc.

Significant correlation in linkage signals from genome-wide scans of schizophrenia and schizotypy

Prior family and adoption studies have suggested a genetic relationship between schizophrenia and schizotypy. However, this has never been verified using linkage methods. We therefore attempted to test for a correlation in linkage signals from genome-wide scans of schizophrenia and schizotypy. The Irish study of high-density schizophrenia families comprises 270 families with at least two members with schizophrenia or poor-outcome schizoaffective disorder (n = 637). Non-psychotic relatives were assessed using the structured interview for schizotypy (n = 746). A 10-cM multipoint, non-parametric, autosomal genomewide scan of schizophrenia was performed in Merlin. A scan of a quantitative trait comprising ratings of DSM-III-R criteria for schizotypal personality disorder in non-psychotic relatives was also performed. Schizotypy logarithm of the odds (LOD) scores were regressed onto schizophrenia LOD scores at all loci, with adjustment for spatial autocorrelation. To assess empirical significance, this was also carried out using 1000 null scans of schizotypy. The number of jointly linked loci in the real data was compared to distribution of jointly linked loci in the null scans. No markers were suggestively linked to schizotypy based on strict Lander Kruglyak criteria. Schizotypy LODs predicted schizophrenia LODs above chance expectation genome wide (empirical P = 0.04). Two and four loci yielded nonparametric LOD (NPLs) > 1.0 and > 0.75, respectively, for both schizophrenia and schizotypy (genome-wide empirical P = 0.04 and 0.02, respectively). These results suggest that at least a subset of schizophrenia susceptibility genes also affects schizotypy in non-psychotic relatives. Power may therefore be increased in molecular genetic studies of schizophrenia if they incorporate measures of schizotypy in non-psychotic relatives.

A molecular model for drug binding to tandem repeats of telomeric G-quadruplexes.

The extreme 3'-ends of human telomeres consist of 150–250 nucleotides of single-stranded DNA sequence together with associated proteins. Small-molecule ligands can compete with these proteins and induce a conformational change in the DNA to a four-stranded quadruplex arrangement, which is also no longer a substrate for the telomerase enzyme. The modified telomere ends provide signals to the DNA-damage-response system and trigger senescence and apoptosis. Experimental structural data are available on such quadruplex complexes comprising up to four telomeric DNA repeats, but not on longer systems that are more directly relevant to the single-stranded overhang in human cells. The present paper reports on a molecular modelling study that uses Molecular Dynamics simulation methods to build dimer and tetramer quadruplex repeats. These incorporate ligand-binding sites and are models for overhang–ligand complexes.

Cell-to-Cell Interactions and Signals Involved in the Reconstitution of Peripheral CD8(+) T-CM and T-EM Cell Pools

We here describe novel aspects of CD8(+) and CD4(+) T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8(+) T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naive CD8(+) T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4(+) T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4(+) T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44(+)CD62L(high)) T-CM and (CD44(+)CD62L(low)) T-EM CD8(+) T cells, we found that the accumulation of TCM and TEM subsets is differentially regulated. T-CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4(+) T-cell help. In contrast, TEM-cell expansion was mainly determined by CD4(+) T-cell help and dependent on the expression of IL-2R beta by CD8 cells, on IL-2 produced by CD4(+) T-cells, on IL-15 and to a minor extent on IL-6.

Current-induced atomic dynamics, instabilities, and Raman signals: Quasiclassical Langevin equation approach

We derive and employ a semiclassical Langevin equation obtained from path integrals to describe the ionic dynamics of a molecular junction in the presence of electrical current. The electronic environment serves as an effective nonequilibrium bath. The bath results in random forces describing Joule heating, current-induced forces including the nonconservative wind force, dissipative frictional forces, and an effective Lorentz-type force due to the Berry phase of the nonequilibrium electrons. Using a generic two-level molecular model, we highlight the importance of both current-induced forces and Joule heating for the stability of the system. We compare the impact of the different forces, and the wide-band approximation for the electronic structure on our result. We examine the current-induced instabilities (excitation of runaway "waterwheel" modes) and investigate the signature of these in the Raman signals.

Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

Simulations of Biased Agonists in the β2 Adrenergic Receptor with Accelerated Molecular Dynamics

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signalling pathway a GPCR promotes intracellular signals though ß-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signalling through the G protein and ß-arrestin. Here we report on the dynamics of the ß2 adrenergic receptor bound to the ß-arrestin and G protein biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring the transition within the nanosecond timescale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the ß-arrestin biased agonist, N-cyclopentylbutanepherine we observe a different pattern of motions in helix 7 when compared to simulations with the G protein biased agonist, Salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs. © 2013 American Chemical Society

A molecular photoionic AND gate based on fluorescent signalling

MOLECULES that perform logic operations are prerequisites for molecular information processing and computation. We and others have previously reported receptor molecules that can be considered to perform simple logic operations by coupling ionic bonding or more complex molecular-recognition processes with photonic (fluorescence) signals: in these systems, chemical binding (the 'input') results in a change in fluorescence intensity (the 'output') from the receptor. Here we describe a receptor (molecule (1) in Fig. 1) that operates as a logic device with two input channels: the fluorescence signal depends on whether the molecule binds hydrogen ions, sodium ions or both. The input/output characteristics of this molecular device correspond to those of an AND gate.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.