Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols1

Tomato (Lycopersicon esculentum) plants were transformed with gene constructs containing a tomato alcohol dehydrogenase (ADH) cDNA (ADH 2) coupled in a sense orientation with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter. Ripening fruit from plants transformed with the constitutively expressed transgene(s) had a range of ADH activities; some plants had no detectable activity, whereas others had significantly higher ADH activity, up to twice that of controls. Transformed plants with fruit-specific expression of the transgene(s) also displayed a range of enhanced ADH activities in the ripening fruit, but no suppression was observed. Modified ADH levels in the ripening fruit influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor production. Hexanol and Z-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity. Concentrations of the respective aldehydes were generally unaltered. The phenotypes of modified fruit ADH activity and volatile abundance were transmitted to second-generation plants in accordance with the patterns of inheritance of the transgenes. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified as having a more intense “ripe fruit” flavor.

Genetic Manipulation of Condensed Tannins in Higher Plants1 : II. Analysis of Birdsfoot Trefoil Plants Harboring Antisense Dihydroflavonol Reductase Constructs

We have produced and analyzed transgenic birdsfoot trefoil (Lotus corniculatus L.) plants harboring antisense dihydroflavonol reductase (AS-DFR) sequences. In initial experiments the effect of introducing three different antisense Antirrhinum majus L. DFR constructs into a single recipient genotype (S50) was assessed. There were no obvious effects on plant biomass, but levels of condensed tannins showed a statistical reduction in leaf, stem, and root tissues of some of the antisense lines. Transformation events were also found, which resulted in increased levels of condensed tannins. In subsequent experiments a detailed study of AS-DFR phenotypes was carried out in genotype S33 using pMAJ2 (an antisense construct comprising the 5′ half of the A. majus cDNA). In this case, reduced tannin levels were found in leaf and stem tissues and in juvenile shoot tissues. Analysis of soluble flavonoids and isoflavonoids in tannin down-regulated shoot tissues indicated few obvious default products. When two S33 AS-DFR lines were outcrossed, there was an underrepresentation of transgene sequences in progeny plants and no examples of inheritance of an antisense phenotype were observed. To our knowledge, this is the first report of the genetic manipulation of condensed tannin biosynthesis in higher plants.

Manipulation of Catalase Levels Produces Altered Photosynthesis in Transgenic Tobacco Plants1

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalytic form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (Γ) at a leaf temperature of 38°C. A significant linear increase was observed in Γ with decreasing catalase (at 50% lower catalase activity Γ increased 39%). There was a significant temperature-dependent linear decrease in Γ in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase Γ decreased 17%). At 29°C, Γ also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38°C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guérin strains that secrete cytokines.

Bacille Calmette-Guérin (BCG) is a live, attenuated strain of Mycobacterium bovis used widely for tuberculosis prophylaxis and bladder cancer immunotherapy, although it has limitations in both contexts. To investigate whether BCG's immunostimulatory properties could be modified, and to gain insight into the interaction between mycobacteria and their hosts, we constructed recombinant BCG strains that secrete functional murine cytokines and studied their properties in mouse models of experimental infection. Cell-mediated immune responses to mycobacterial antigen (purified protein derivative) were assayed using splenocytes from mice inoculated with various BCG recombinants. Antigen-specific proliferation and cytokine release were found to be substantially greater with splenocytes derived from mice injected with cytokine-secreting BCG than with splenocytes from mice injected with BCG lacking cytokines. The most profound effects were induced by BCG secreting interleukin 2, interferon gamma, or granulocyte-macrophage colony-stimulating factor. Thus, cytokine-secreting BCG can enhance immune responses to mycobacterial antigens and may be improved reagents for tuberculosis prophylaxis and cancer immunotherapy.

Generalized isotropic Lipkin-Meshkov-Glick models: ground state entanglement and quantum entropies

We introduce a new class of generalized isotropic Lipkin–Meshkov–Glick models with su(m+1) spin and long-range non-constant interactions, whose non-degenerate ground state is a Dicke state of su(m+1) type. We evaluate in closed form the reduced density matrix of a block of Lspins when the whole system is in its ground state, and study the corresponding von Neumann and Rényi entanglement entropies in the thermodynamic limit. We show that both of these entropies scale as a log L when L tends to infinity, where the coefficient a is equal to (m  −  k)/2 in the ground state phase with k vanishing magnon densities. In particular, our results show that none of these generalized Lipkin–Meshkov–Glick models are critical, since when L-->∞ their Rényi entropy R_q becomes independent of the parameter q. We have also computed the Tsallis entanglement entropy of the ground state of these generalized su(m+1) Lipkin–Meshkov–Glick models, finding that it can be made extensive by an appropriate choice of its parameter only when m-k≥3. Finally, in the su(3) case we construct in detail the phase diagram of the ground state in parameter space, showing that it is determined in a simple way by the weights of the fundamental representation of su(3). This is also true in the su(m+1) case; for instance, we prove that the region for which all the magnon densities are non-vanishing is an (m  +  1)-simplex in R^m whose vertices are the weights of the fundamental representation of su(m+1).

Control framework for dexterous manipulation using dynamic visual servoing and tactile sensors’ feedback

Tactile sensors play an important role in robotics manipulation to perform dexterous and complex tasks. This paper presents a novel control framework to perform dexterous manipulation with multi-fingered robotic hands using feedback data from tactile and visual sensors. This control framework permits the definition of new visual controllers which allow the path tracking of the object motion taking into account both the dynamics model of the robot hand and the grasping force of the fingertips under a hybrid control scheme. In addition, the proposed general method employs optimal control to obtain the desired behaviour in the joint space of the fingers based on an indicated cost function which determines how the control effort is distributed over the joints of the robotic hand. Finally, authors show experimental verifications on a real robotic manipulation system for some of the controllers derived from the control framework.

Visual perception for the 3D recognition of geometric pieces in robotic manipulation

During grasping and intelligent robotic manipulation tasks, the camera position relative to the scene changes dramatically because the robot is moving to adapt its path and correctly grasp objects. This is because the camera is mounted at the robot effector. For this reason, in this type of environment, a visual recognition system must be implemented to recognize and “automatically and autonomously” obtain the positions of objects in the scene. Furthermore, in industrial environments, all objects that are manipulated by robots are made of the same material and cannot be differentiated by features such as texture or color. In this work, first, a study and analysis of 3D recognition descriptors has been completed for application in these environments. Second, a visual recognition system designed from specific distributed client-server architecture has been proposed to be applied in the recognition process of industrial objects without these appearance features. Our system has been implemented to overcome problems of recognition when the objects can only be recognized by geometric shape and the simplicity of shapes could create ambiguity. Finally, some real tests are performed and illustrated to verify the satisfactory performance of the proposed system.

THE DARK PERSONALITY AND JOB SUCCESS IN THE UNITED STATES ARMY: USE OF INTERPERSONAL MANIPULATION IN THE WORKPLACE

This dissertation relates job desires and outcomes to the Dark Personality (Psychopathy, Machiavellianism, Narcissism, Low Agreeableness, Low Honesty-Humility) in the United States Army. It purports that individuals high on the Dark Personality desire more power, money, and status, and that they obtain jobs that afford them these luxuries by using manipulation at work. Two pilot studies used samples of United States Army members to create and test index variables: Dark Personality, Total Manipulation in the workplace, Desire for Job Success, and Total Job Success in the Army. Individual personality traits, manipulation tactics, and job desires were examined in secondary analyses. Using a sample of 468 United States Army Members, central analyses indicated that Army members high on the Dark Personality desired Job Success. Likewise, army members higher on the Dark Personality used more Manipulation tactics at work, including the egregious tactics. Yet, using more Manipulation tactics at work predicted lower levels of Job Success in the Army. Most manipulation tactics had a negative impact on Job Success, with the exception of soft tactics like Reason and Responsibility Invocation. Together, these results indicate that selective use of soft manipulation predicted Job Success, but use of more manipulation tactics predicted less Job Success in the Army. Curvilinear results indicated that being either very low or very high on the Dark Personality predicted more Job Success in the Army, whereas having intermediate levels of the Dark Personality predicted less Job Success. Finally, possessing the Dark Personality and using more Manipulation tactics at work, together, predicted less Job Success in the Army. Collectively, the results indicate that army members with intermediate levels of the Dark Personality want more powerful and high paying jobs, yet their strategy of manipulating their coworkers to move up the job ladder does not result in higher ranking, higher paying Army positions. However, Army members highest on the Dark Personality achieved job success, defying the maladaptive influence that antisocial personality traits and manipulative behaviour had on job success for most Army members. Therefore, this dissertation indicates that successful corporate scoundrels exist in the Army, but there are few of them.

The New EU Criminal Law Competence in Action: The Proposal for a Directive on Criminal Sanctions for Insider Dealing and Market Manipulation. IES WORKING PAPER 5/2013

The aim of this paper is to analyse the proposed Directive on criminal sanctions for insider dealing and market manipulation (COM(2011)654 final), which represents the first exercise of the European Union competence provided for by Article 83(2) of the Treaty on the Functioning of the European Union. The proposal aims at harmonising the sanctioning regimes provided by the Member States for market abuse, imposing the introduction of criminal sanctions and providing an opportunity to critically reflect on the position taken by the Commission towards the use of criminal law. The paper will discuss briefly the evolution of the EU’s criminal law competence, focusing on the Lisbon Treaty. It will analyse the ‘essentiality standard’ for the harmonisation of criminal law included in Article 83(2) TFEU, concluding that this standard encompasses both the subsidiarity and the ultima ratio principles and implies important practical consequences for the Union’s legislator. The research will then focus on the proposed Directive, trying to assess if the Union’s legislator, notwithstanding the ‘symbolic’ function of this proposal in the financial crisis, provides consistent arguments on the respect of the ‘essentiality standard’. The paper will note that the proposal raises some concerns, because of the lack of a clear reliance on empirical data regarding the essential need for the introduction of criminal law provisions. It will be stressed that only the assessment of the essential need of an EU action, according to the standard set in Article 83(2) TFEU, can guarantee a coherent choice of the areas interested by the harmonisation process, preventing the legislator to choose on the basis of other grounds.