Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Arabidopsis Rho-Related GTPases: Differential Gene Expression in Pollen and Polar Localization in Fission Yeast1

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

The notion of the Cambrian pananimalia genome.

The toil by photosynthesizing cyanobacteria and blue-green algae of nearly three billion years appeared to have finally resulted in the sufficient accumulation of molecular oxygen. So, the stage was set for the emergence, at the ocean bottom, of diverse animals that were consumers of molecular oxygen. It now appears that this Cambrian explosion, during which nearly all the extant animal phyla have emerged, was of an astonishingly short duration, lasting only 6-10 million years. Inasmuch as only a 1% DNA base sequence change is expected in 10 million years under the standard spontaneous mutation rate, I propose that all those diverse animals of the early Cambrian period, some 550 million years ago, were endowed with nearly identical genomes, with differential usage of the same set of genes accounting for the extreme diversities of body forms. Some of the more pertinent genes that are thought to be included in the Cambrian pananimalia genome are as follows. (i) A gene for lysyloxidase that, in the presence of molecular oxygen, crosslinked collagen triple helices to produce ligaments and tendons, thus contributing to the stout bodies of the Cambrian animals. (ii) Genes for hemoglobin; these internal transporters of molecular oxygen are today seen sporadically in members of diverse animal phyla. (iii) The Pax-6 gene for eye formation; the eyes of a ribbon worm to a human are organized by this gene. In animals without eyes, the same gene organizes other sensory systems and organs. (iv) A series of Hox genes for the anterior-posterior (cranio-caudal) body plans: these genes are also present in all phyla of the kingdom Animalia.

Relationship between taxocenoses of decapod crustaceans and characteristics of coastal detritic bottoms in the east and southeast of the Spanish coast

Se ha estudiado la composición de las poblaciones de crustáceos decápodos en los fondos detríticos circalitorales del Este y Sureste ibérico, con relación a sus diferentes facies, efectuándose diferentes análisis estadísticos sobre la composición y diversidad. La taxocenosis de crustáceos decápodos muestra elevados valores de abundancia, riqueza y diversidad en las localidades identificadas como fondos detríticos costeros de aspecto típico. Por último, algunos grupos de especies podrían ser utilizados para caracterizar las biocenosis típica de fondos detríticos costeros, tales como: Paguristes eremita, Ebalia edwardsi, E. deshayesi, Eurynome aspera, Galathea intermedia, Parthenope massena, y Anapagurus hyndmani; la biocenosis del detrítico costero enfangada: Ebalia tuberosa, Atelecyclus rotundatus, Ethusa mascarone, y Liocarcinus zariquieyi; y la biocenosis de arenas fangosas: Upogebia deltaura, Goneplax rhomboides.

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.

III. Harvard College Papers, 1st series, Volume 3, 1785-1796

The third volume of the College Papers contains original documents dating from 1785 to 1796, spanning the tenures of president Joseph Willard and treasurer Ebenezer Storer. The series chiefly consists of incoming correspondence for Storer.

III. Harvard College Papers, 1st series, Volume 3, 1785-1796

The third volume of the College Papers contains original documents dating from 1785 to 1796, spanning the tenures of president Joseph Willard and treasurer Ebenezer Storer. The series chiefly consists of incoming correspondence for Storer.

Letters from William Tudor to John Thornton Kirkland,

Four letters written from Boston regarding plans to establish a new literary periodical, the North American Review. Tudor asks Kirkland to contribute to the periodical and describes plans to establish a lecture series at the Boston Athenaum.

A proof of Jesus Christ's being the ancient promised Messiah: a sermon preached in the Chapel of Harvard College at Cambridge in New England, June 23, 1756

The hand-sewn notebook contains a 24-page manuscript draft of the second Dudleian lecture, delivered by John Barnard on June 23, 1756. The copy includes a small number of edits and struck-out words adopted in the printed version published by J. Draper of Boston in 1756. The sermon begins with the Biblical text Mark 14:61, 62. The covers are no longer with the item.

A discourse on natural religion: delivered in the Chapel of the University at Cambridge, September 5, 1787

The hand-sewn notebook contains a 35-page manuscript draft of the Dudleian lecture delivered by Simeon Howard on September 5, 1787 at Harvard College. The sermon begins with the Biblical text Acts 17:28. The copy includes a small number of edits and struck-out words. The covers are no longer with the item. The lecture was not printed.

EEAS 2.0. A legal commentary on Council Decision 2010/427/EU establishing the organisation and functioning of the European External Action Service. EIPA Working Paper, 2013

From the Preface. Pursuant to Article 13(3) of Council Decision 2010/427/EU of 26 July 2010 establishing the organisation and functioning of the European External Action Service, the High Representative is held to provide a review of the organisation and functioning of the EEAS by mid-­‐2013. This review will cover, inter alia, the implementation of Article 6(6), (8) and (11), so as to ensure an adequate geographical and gender balance and a meaningful presence of nationals from all member states in the EEAS. If necessary, the review will be accompanied by appropriate proposals for the revision of the 2010 Council Decision (e.g., suggestions for additional specific measures to correct possible imbalances of staffing). In that case, the Council will, in accordance with Article 27(3) TEU, revise the Decision in light of the review by the beginning of 2014. This short and user-­‐friendly legal commentary on the 2010 Council Decision is the first of its kind and is intended to inform those involved in the review process and to serve as a reference document for practitioners and analysts dealing with the EEAS. This commentary is not an elaborate doctrinal piece, but rather a textual and contextual analysis of each article, that takes account of i) other relevant legal provisions (primary, secondary, international), ii) the process leading to the adoption of the 2010 Council Decision (i.e. travauxpréparatoires), iii) the preamble of the Council Decision, and iv) insofar as it is possible at this stage, early implementation. Wherever relevant, cross-­‐references to other provisions of the EEAS Council Decision have been made so as to tie in the different commentaries and ensure overall consistency.

Differentiated Integration in the EU - From the inside looking out. CEPS Paperbacks. January 2014

Flexibility might be convenient when it comes to accommodating member states’ differing socio-economic and political interests in an expanding EU. Yet, opt-outs, enhanced cooperation and cooperation between member states outside the EU’s legal framework are calling into question the boundaries of this constitutional, institutional and legal differentiation in an EU that is founded on common principles, with a specific legal order and common institutions.

The ECB AQR and the EBA Stress Test: What will the numbers tell? CEPS Commentary, 23 October 2014

From the Introduction. With the results of its asset quality review (AQR), to be published on 26 October 2014, the European Central Bank intends to provide clarity on the shape of the 120 banks it will supervise in the eurozone, and it may request a series of follow-up actions before assuming its new set of tasks under the Single Supervisory Mechanism (SSM) Regulation in November. On the same day, the European Banking Authority (EBA) will also be publishing the results of its stress test, covering 123 banks across 22 European Economic Area (EEA) countries. For the ECB, it will be a matter of setting the standard for its future task, whereas EBA, seeks to restore the confidence it lost in the 2011 stress test and 2012 capital exercise. Both institutions will need to indicate how they will cooperate in the future in these tasks, and through enhanced disclosure, strengthen the confidence in the European banking system.

The EU’s Response to the Refugee Crisis: Taking Stock and Setting Policy Priorities. CEPS Essay No. 20, 16 December 2015

What have been the most important EU policy and legal responses to the 2015 refugee crisis? Is Europe taking effectively responsibility in compliance with its founding principles? This Essay takes stock of the main results and policy outputs from the EU’s interventions in the refugee crisis. It critically highlights the outstanding policy dilemmas confronting the adopted instruments and puts forwards a set of policy priorities to guide the next phases of the European Agenda on Migration.