The Cosmological Constant Problems

The old cosmological constant problem is to understand why the vacuum energy is so small; the new problem is to understand why it is comparable to the present mass density. Several approaches to these problems are reviewed. Quintessence does not help with either; anthropic considerations offer a possibility of solving both. In theories with a scalar field that takes random initial values, the anthropic principle may apply to the cosmological constant, but probably to nothing else

Dysregulated RasGRP1 Responds to Cytokine Receptor Input in T Cell Leukemogenesis

Enhanced signaling by the small guanosine triphosphatase Ras is common in T cell acute lymphoblastic leukemia/lymphoma (T-ALL), but the underlying mechanisms are unclear. We identified the guanine nucleotide exchange factor RasGRP1 (Rasgrp1 in mice) as a Ras activator that contributes to leukemogenesis. We found increased RasGRP1 expression in many pediatric T-ALL patients, which is not observed in rare early T cell precursor T-ALL patients with KRAS and NRAS mutations, such as K-Ras[superscript G12D]. Leukemia screens in wild-type mice, but not in mice expressing the mutant K-Ras[superscript G12D] that encodes a constitutively active Ras, yielded frequent retroviral insertions that led to increased Rasgrp1 expression. Rasgrp1 and oncogenic K-Ras[superscript G12D] promoted T-ALL through distinct mechanisms. In K-Ras[superscript G12D] T-ALLs, enhanced Ras activation had to be uncoupled from cell cycle arrest to promote cell proliferation. In mouse T-ALL cells with increased Rasgrp1 expression, we found that Rasgrp1 contributed to a previously uncharacterized cytokine receptor–activated Ras pathway that stimulated the proliferation of T-ALL cells in vivo, which was accompanied by dynamic patterns of activation of effector kinases downstream of Ras in individual T-ALLs. Reduction of Rasgrp1 abundance reduced cytokine-stimulated Ras signaling and decreased the proliferation of T-ALL in vivo. The position of RasGRP1 downstream of cytokine receptors as well as the different clinical outcomes that we observed as a function of RasGRP1 abundance make RasGRP1 an attractive future stratification marker for T-ALL.National Institutes of Health (U.S.). Pioneer AwardNational Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874)National Institutes of Health (U.S.). (P01 AI091580

Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis

Peer reviewedPublisher PD

Characterization of the Promoter, MxiE Box and 5′ UTR of Genes Controlled by the Activity of the Type III Secretion Apparatus in Shigella flexneri

Activation of the type III secretion apparatus (T3SA) of Shigella flexneri, upon contact of the bacteria with host cells, and its deregulation, as in ipaB mutants, specifically increases transcription of a set of effector-encoding genes controlled by MxiE, an activator of the AraC family, and IpgC, the chaperone of the IpaB and IpaC translocators. Thirteen genes carried by the virulence plasmid (ospB, ospC1, ospD2, ospD3, ospE1, ospE2, ospF, ospG, virA, ipaH1.4, ipaH4.5, ipaH7.8 and ipaH9.8) and five genes carried by the chromosome (ipaHa-e) are regulated by the T3SA activity. A conserved 17-bp MxiE box is present 5′ of most of these genes. To characterize the promoter activity of these MxiE box-containing regions, similar ∼67-bp DNA fragments encompassing the MxiE box of 14 MxiE-regulated genes were cloned 5′ of lacZ in a promoter probe plasmid; β-galactosidase activity detected in wild-type and ipaB strains harboring these plasmids indicated that most MxiE box-carrying regions contain a promoter regulated by the T3SA activity and that the relative strengths of these promoters cover an eight-fold range. The various MxiE boxes exhibiting up to three differences as compared to the MxiE box consensus sequence were introduced into the ipaH9.8 promoter without affecting its activity, suggesting that they are equally efficient in promoter activation. In contrast, all nucleotides conserved among MxiE boxes were found to be involved in MxiE-dependent promoter activity. In addition, we present evidence that the 5′ UTRs of four MxiE-regulated genes enhance expression of the downstream gene, presumably by preventing degradation of the mRNA, and the 5′ UTRs of two other genes carry an ancillary promoter

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

AIMS/HYPOTHESIS Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. METHODS Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. RESULTS Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. CONCLUSIONS/INTERPRETATION Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects

Evidence for Epithelial-Mesenchymal Transition in Cancer Stem Cells of Head and Neck Squamous Cell Carcinoma

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1+ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1+ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44+/CD24− was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44+/CD24−/ALDH1+ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis

Neuropeptide Signaling Differentially Affects Phase Maintenance and Rhythm Generation in SCN and Extra-SCN Circadian Oscillators

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC2, play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC2 receptors (Vipr2−/−) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC2 receptor expression; both VPAC2 receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC2 receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2−/− mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC2 receptor. Vipr2−/− SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2−/− Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2−/− cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2−/− mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2−/− animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting

RAD21 Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21 - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved