Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

A novel role for neural crest cells in thymus organogenesis

Classical ablation studies have shown that neural crest cells (NCC) are critical for thymus organogenesis, though their role in this process has never been determined. We have used a mouse model deficient in NCC near the thymus rudiment to investigate the role of NCC in thymus organogenesis. Splotch mice exhibit a lack of NCC migration due to mutation in the gene encoding the transcription factor Pax 3. Homozygous mutants, designated Pax3Sp/Sp, display a range of phenotypes including spina bifida, cardiac outflow tract deformities, and craniofacial deformities. Pax3Sp/Sp, mice have also been reported to have hypoplastic and abnormal thymi, which is consistent with the expected result based on the classical ablation studies. However, in contrast to the dogma, we find that the thymus lobes in Pax3Sp/Sp, mice are even larger in size than those of littermate controls, although they fail to migrate and are therefore ectopic. Differentiation of the thymic epithelial compartments occurs normally, including the ability to import hematopoietic precursors, until the embryos die at embryonic day E13.0. We also investigated the patterning of the third pharyngeal pouch which gives rise to both the thymus and the parathyroid. Using RNA probes to detect expression of transcription factors exclusively expressed in the ventral, thymus- or dorsal, parathyroidfated domains of the E11.5 third pouch, we show that the parathyroid domain is restricted and the thymus-fated domain is expanded in Pax3Sp/Sp, embryos. Furthermore, mixing of the boundary between these domains occurs at E12.0. These results necessitate reconsideration of the previously accepted role for NCC in thymus organogenesis. NCC are not required for outgrowth of the thymus up to E13.0, and most strikingly, we have discovered a novel role for NCC in establishing parathyroid versus thymus fate boundaries in the third pharyngeal pouch. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^

Latin American women's experiences with medical abortion in settings where abortion is legally restricted.

El aborto está legalmente restringido en la mayoría de América Latina donde 95 % de los 4.4 millones de abortos que se realizan anualmente son inseguros. El aborto con medicamentos es el uso de una droga o una combinación de drogas para interrupir el embarazo. Mifepristona seguida de misoprostol constituye el regimen más efectivo y recomendado. En los lugares donde no está disponible la mifepristona, se utiiza misoprostol solo. El aborto con medicamentos ha transformado radicalmente la práctica del aborto a nivel mundial, y particularmente en los contextos legalmente restrictivos. En América Latina, desde hace más de dos décadas, las mujeres utilizan el misoprostol para autoinducirse abortos. Este artículo resume los hallazgos de una revisión bibliográfica sobre las experiencias de las mujeres con el aborto con medicamentos en países latinoamericanos donde el aborto voluntario es ilegal. Las experiencias personales de las mujeres con el aborto con medicamentos son diversas y varían según el contexto, la edad, la historia reproductiva, el nivel socioeconómico y el conocimiento acerca del aborto con medicamentos así como las circunstancias físicas, emocionales y sociales que rodean el embarazo. Pero fundamentalmente, las experiencias están determinadas por la posibilidad de las mujeres de acceder a: 1) un aborto clandestino realizado bajo supervisión médica, o 2) información completa y precisa acerca del aborto con medicamentos, Otros factores clave incluyen el acceso a recursos económicos y apoyo emocional. Las mujeres valoran la seguridad y efectividad del aborto con medicamentos así como la privacidad que ofrece y la posibilidad de tener cerca a su pareja, amiga/o, o persona de su confianza durante el proceso. Las mujeres perciben al aborto con medicamentos como menos doloroso, más fácil, más seguro, más práctico, menos costoso, más natural y menos traumático que otros métodos abortivos. Que sea auto-inducido y que evite el procedimiento quirúrgico también son señalados como ventajas. Las principales desventajas identificadas son que es doloroso y que lleva tiempo para que se complete. Otros aspectos evaluados negativamente incluyen los efectos secundarios, el sangrado prolongado, la posibilidad de que no sea efectivo, y el hecho de que algunas mujeres eventualmente deban solicitar atención médica en una institución donde sean sancionadas por haberse practicado un aborto y hasta denunciadas a la policía.

Women’s experiences with the use of medical abortion in a legally restricted context: the case of Argentina

This article presents the findings of a qualitative study exploring the experiences of women living in Buenos Aires Metropolitan Area, Argentina, with the use of misoprostol for inducing an abortion. We asked women about the range of decisions they had to make, their emotions, the physical experience, strategies they needed to use, including seeking health care advice and in dealing with a clandestine medical abortion, and their overall evaluation of the experience. An in-depth interview schedule was used. The women had either used misoprostol and sought counselling or care at a public hospital (n=24) or had used misoprostol based on the advice of a local hotline, information from the internet or from other women (n=21). Four stages in the women’s experiences were identified: how the decision to terminate the pregnancy was taken, how the medication was obtained, how the tablets were used, and reflections on the outcome whether or not they sought medical advice. Safety and privacy were key in deciding to use medical abortion. Access to the medication was the main obstacle, requiring a prescription or a friendly drugstore. Correct information about the number of pills to use and dosage intervals was the least easy to obtain and caused concerns. The possibility of choosing a time of privacy and having the company of a close one was highlighted as a unique advantage of medical abortion. Efforts to improve abortion law, policy and service provision in Argentina in order to ensure the best possible conditions for use of medical abortion by women should be redoubled.

Isotopic composition of rocks from the Lau/Tonga back-arc basin, data of DSDP Leg 21 and ODP Legs 91 an 135

New trace element, Sr-, Nd-, Pb- and Hf isotope data provide insights into the evolution of the Tonga-Lau Basin subduction system. The involvement of two separate mantle domains, namely Pacific MORB mantle in the pre-rift and early stages of back-arc basin formation, and Indian MORB mantle in the later stages, is confirmed by these results. Contrary to models proposed in recent studies on the basis of Pb isotope and other compositional data, this change in mantle wedge character best explains the shift in the isotopic composition, particularly 143Nd/144Nd ratios, of modern Tofua Arc magmas relative to all other arc products from this region. Nevertheless, significant changes in the slab-derived flux during the evolution of the arc system are also required to explain second order variations in magma chemistry. In this region, the slab-derived flux is dominated by fluid; however, these fluids carry Pb with sediment-influenced isotopic signatures, indicating that their source is not restricted to the subducting altered mafic oceanic crust. This has been the case from the earliest magmatic activity in the arc (Eocene) until the present time, with the exception of two periods of magmatic activity recorded in samples from the Lau Islands. Both the Lau Volcanic Group, and Korobasaga Volcanic Group lavas preserve trace element and isotope evidence for a contribution from subducted sediment that was not transported as a fluid, but possibly in the form of a melt. This component shares similarities with that influencing the chemistry of the northern Tofua Arc magmas, suggesting some caution may be required in the adoption of constraints for the latter dependent upon the involvement of sediments from the Louisville Ridge. A key outcome of this study is to demonstrate that the models proposed to explain subduction zone magmatism cannot afford to ignore the small but important contributions made by the mantle wedge to the incompatible trace element inventory of arc magmas.

Climate sensitivity across marine domains of life: limits to evolutionary adaptation shape species interactions, supplementary material

Organisms in all domains, Archaea, Bacteria, and Eukarya will respond to climate change with differential vulnerabilities resulting in shifts in species distribution, coexistence, and interactions. The identification of unifying principles of organism functioning across all domains would facilitate a cause and effect understanding of such changes and their implications for ecosystem shifts. For example, the functional specialization of all organisms in limited temperature ranges leads us to ask for unifying functional reasons. Organisms also specialize in either anoxic or various oxygen ranges, with animals and plants depending on high oxygen levels. Here, we identify thermal ranges, heat limits of growth, and critically low (hypoxic) oxygen concentrations as proxies of tolerance in a meta-analysis of data available for marine organisms, with special reference to domain-specific limits. For an explanation of the patterns and differences observed, we define and quantify a proxy for organismic complexity across species from all domains. Rising complexity causes heat (and hypoxia) tolerances to decrease from Archaea to Bacteria to uni- and then multicellular Eukarya. Within and across domains, taxon-specific tolerance limits likely reflect ultimate evolutionary limits of its species to acclimatization and adaptation. We hypothesize that rising taxon-specific complexities in structure and function constrain organisms to narrower environmental ranges. Low complexity as in Archaea and some Bacteria provide life options in extreme environments. In the warmest oceans, temperature maxima reach and will surpass the permanent limits to the existence of multicellular animals, plants and unicellular phytoplankter. Smaller, less complex unicellular Eukarya, Bacteria, and Archaea will thus benefit and predominate even more in a future, warmer, and hypoxic ocean.

Appendix 7: List of species with their distribution ranges restricted to the corresponding phytogeographical region

Understanding species distribution patterns and the corresponding environmental determinants is a crucial step in the development of effective strategies for the conservation and management of plant communities and ecosystems. Therefore, a central prerequisite is the biogeographical and macroecological analysis of factors and processes that determine contemporary, potential, as well as future geographic distribution of species. This thesis has been conducted in the framework of the BIOMAPS-BIOTA project at the Nees Institute of Biodiversity of Plants, which was funded by the German Federal Ministry of Education and Research (BMBF). The study investigated patterns of plants species richness and phytogeographic regions under contemporary environmental conditions and forecasted future climate change in the area of West Africa covering five countries: Benin, Burkina Faso, Côte d'Ivoire, Ghana and Togo. Firstly, geographic patterns of vascular plant species richness have been depicted at a relatively fine spatial resolution based on the potential distribution of 3,393 species. Species richness is closely related to the steep climatic gradient existing in the region with a high concentration of species in the most humid areas in the south and decreases towards the northern drier areas. The investigation of the effectiveness of the existing network of protected areas shows an overall good coverage of species in the study area. However, the proportion of covered species is considerably lower at national extent for some countries, thus calling for more protected areas in order to cover adequately a maximum number of plants species in these countries. Secondly, based on the potential distribution range of vascular plant species, seven phytogeographic regions have been delineated that broadly reflect the vegetation zones as defined by White (1983). However notable differences to the delineation of White (1983) occur at the margins of some regions. Corresponding to a general southward shifted of all regions. And expansion of the Sahel vegetation zone is observed in the north, while the rainforest zone is decreased in the very south.This is alarming since the rainforest shelters a high number of species and a high proportion of range-restricted or endemic species, despite their relatively small extent compared to the other regions. Finally, the evaluation of the potential impact of climate change on plant species richness in the study area, results in a severe loss of future suitable habitat for up to 50% of species per grid cell, particularly in the rainforest region. Moreover, the analysis of the possible shift of phytogeographic regions shows in general a strong deterioration of the West African rainforest. In contrast the drier areas are expanding continuously, although a slight gain in species number can be observed in some particular regions. The overall lesson to retain from the results of this study is that the West African rainforest should be fixed as a high priority area for the conservation of biodiversity of plants, since it is subject to severe contemporary and projected future threats.

Inherited structural controls on fault geometry, architecture and hydrothermal activity: an example from Grimsel Pass, Switzerland

Exhumed faults hosting hydrothermal systems provide direct insight into relationships between faulting and fluid flow, which in turn are valuable for making hydrogeological predictions in blind settings. The Grimsel Breccia Fault (Aar massif, Central Swiss Alps) is a late Neogene, exhumed dextral strike-slip fault with a maximum displacement of 25–45 m, and is associated with both fossil and active hydrothermal circulation. We mapped the fault system and modelled it in three dimensions, using the distinctive hydrothermal mineralisation as well as active thermal fluid discharge (the highest elevation documented in the Alps) to reveal the structural controls on fluid pathway extent and morphology. With progressive uplift and cooling, brittle deformation inherited the mylonitic shear zone network at Grimsel Pass; preconditioning fault geometry into segmented brittle reactivations of ductile shear zones and brittle inter-shear zone linkages. We describe ‘pipe’-like, vertically oriented fluid pathways: (1) within brittle fault linkage zones and (2) through alongstrike- restricted segments of formerly ductile shear zones reactivated by brittle deformation. In both cases, low-permeability mylonitic shear zones that escaped brittle reactivation provide important hydraulic seals. These observations show that fluid flow along brittle fault planes is not planar, but rather highly channelised into sub-vertical flow domains, with important implications for the exploration and exploitation of geothermal energy.

Computational Schemes for Optimizing Domains of Attraction in Dynamical Systems

In this paper, several computational schemes are presented for the optimal tuning of the global behavior of nonlinear dynamical sys- tems. Specifically, the maximization of the size of domains of attraction associated with invariants in parametrized dynamical sys- tems is addressed. Cell Mapping (CM) tech- niques are used to estimate the size of the domains, and such size is then maximized via different optimization tools. First, a ge- netic algorithm is tested whose performance shows to be good for determining global maxima at the expense of high computa- tional cost. Secondly, an iterative scheme based on a Stochastic Approximation proce- dure (the Kiefer-Wolfowitz algorithm) is eval- uated showing acceptable performance at low cost. Finally, several schemes combining neu- ral network based estimations and optimiza- tion procedures are addressed with promising results. The performance of the methods is illus- trated with two applications: first on the well-known van der Pol equation with stan- dard parametrization, and second the tuning of a controller for saturated systems.