Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing

Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3’-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring site for disulfide- and thiol-maleimide conjugate formation, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing

Converse effect of pressure on the quadrupolar and magnetic transition in Ce3Pd20Si6

The heavy fermion compound Ce3Pd20Si6 displays unconventional quantum criticality as the lower of two consecutive phase transitions is fully suppressed by magnetic field. Here we report on the effects of pressure as an additional tuning parameter. Specific heat and electrical resistivity measurements reveal a converse effect of pressure on the two transitions, leading to the merging of both transitions at 6.2 kbars. The field-induced quantum criticality is robust under pressure tuning. We rationalize our findings within an extended version of the global phase diagram for antiferromagnetic heavy fermion quantum criticality

The work of the audience: visual matrix methodology in museums

Visual matrix methodology has been designed for researching cultural imaginaries. It is an image-led, group-based method that creates a “third space” research setting to observe audience groups re-enacting lived experience of an event or process that takes place in the third space of a cultural setting. In this article the method is described through its use in relation to an art-science exhibition, Human + Future of the species, where three audience groups with investments in technology worked with exhibition material to achieve a complex, ambivalent state of mind regarding technological futures. The visual matrix has been designed to capture the affective and aesthetic quality of audience engagement in third space by showing what audiences do with what is presented to them. We argue that such methodologies are useful for museums as they grapple with their role as sites where citizens not only engage in dialogue with one another but actively re-work their imaginaries of the future

Hermeneutics and Nature

This paper contributes to the on-going research into the ways in which the humanities transformed the natural sciences in the late Eighteenth and early Nineteenth Centuries. By investigating the relationship between hermeneutics -- as developed by Herder -- and natural history, it shows how the methods used for the study of literary and artistic works played a crucial role in the emergence of key natural-scientific fields, including geography and ecology

Liposome Mediated-CYP1A1 Gene Silencing Nanomedicine Prepared Using Lipid Film-Coated Proliposomes as a Potential Treatment Strategy of Lung Cancer

The occurrence of lung cancer is linked with tobacco smoking, mainly through the generation of polycyclic aromatic hydrocarbons (PAHs). Elevated activity of cytochrome P4501A1 (CYP1A1) plays an important role in the metabolic processing of PAHs and its carcinogenicity. The present work aimed to investigate the role of CYP1A1 gene in PAH-mediated growth and tumor development in vitro and using an in vivo animal model. RNAi strategy was utilized to inhibit the overexpression of CYP1A1 gene using cationic liposomes generated using a lipid film-coated proliposome microparticles. Treatment of PAH-induced human alveolar adenocarcinoma cell line with cationic liposomes carrying CYP1A1 siRNA resulted in down regulation of CYP1A1 mRNA, protein as well as its enzymatic activity, triggering apoptosis and inhibiting multicellular tumor spheroids formation in vitro. Furthermore, silencing of CYP1A1 gene in BALB/c nude xenografts inhibited tumor growth via down regulation of CYP1A1 expression. Altogether, our findings showed that liposome-based gene delivery technology is a viable and stable approach for targeting cancer causing genes such as CY1PA1. This technology facilitated by the use of sugar particles coated with lipid films has demonstrated ability to generate anticancer effects that might be used in the future for therapeutic intervention and treatment of lung cancer. [Abstract copyright: Copyright © 2019. Published by Elsevier B.V.

Partnerorientierung zwischen Realität und Imagination: Anmerkungen zu einem zentralen Konzept der Dialogtheorie

This paper attempts a critique of the notion of 'dialogue' in dialogue theory as espoused by Linell, Markova, and others building on Bakhtin’s writings. According to them, human communication, culture, language, and even cognition are dialogical in nature. This implies that these domains work by principles of other-orientation and interaction. In our paper, we reject accepting other-orientation as an a priori condition of every semiotic action. Instead, we claim that in order to be an empirically useful concept for the social sciences, it must be shown if and how observable action is other-oriented. This leads us to the following questions: how can we methodically account for other-orientation of semiotic action? Does other-orientation always imply interaction? Is every human expression oriented towards others? How does the other, as s/he is represented in semiotic action, relate to the properties which the other can be seen to exhibit as indexed by their observable behavior? We study these questions by asking how the orientation towards others becomes evident in different forms of communication. For this concern, we introduce ‘recipient design’, ‘positioning’ and ‘intersubjectivity’ as concepts which allow us to inquire how semiotic action both takes the other into account and, reflexively, shapes him/her as an addressee having certain properties. We then specifically focus on actions and situations in which other-orientation is particularly problematic, such as interactions with children, animals, machines, or communication with unknown recipients via mass media. These borderline cases are scrutinized in order to delineate both limits and constitutive properties of other-orientation. We show that there are varieties of meaningful actions which do not exhibit an orientation towards the other, which do not rest on (the possibility of) interaction with the other or which even disregard what their producer can be taken to know about the other. Available knowledge about the other may be ignored in order to reach interactional goals, e. g. in strategical interactions or for concerns of socialization. If semiotic action is otherorientated, its design depends on how the other is available to and matters for their producer. Other-orientation may build on shared biographical experiences with the other, knowledge about the other as an individual and close attention to their situated conduct. However, other-orientation may also rest on (stereo-)typification with respect to institutional roles or group membership. In any case, others as they are represented in semiotic action can never be just others-as-such, but only othersas-perceived-by-the-actor. We conclude that the strong emphasis which dialogue theories put on otherorientation obscures that other-orientation is neither universal in semiotic action, that it must be distinguished from an interactive relationship, and that the ways in which the other figures in semiotic actions is not homogeneous in any of its most general properties. Instead, there is a huge variation in the ways in which the other can be taken into account. Therefore close scrutiny of how the other precisely figures in a certain kind of semiotic action is needed in order to lend the concept of ‘other-orientation’ empirical substance and a definite sense

The Visual Matrix method in a study of death and dying: Methodological reflections

The Visual Matrix method is designed to elicit imagistic and associative contributions established collectively amongst participants in a group setting. In this article, a hard to-reach area of experience - death and dying - illustrates the production of shared cultural images beyond individual experience. Our dual purpose was to assess the suitability of the method for this challenging topic, and to understand the ways in which death figured in the imagination of the participants. Three theorists, Wilfred Bion, Alfred Lorenzer and Gilles Deleuze, enable us to theorise psychosocial processes of symbolisation beyond cognition