Fully Reprogrammable 2D Array of Multi-State Molecular Switching Units.

Integration of molecular switching units into complex electronic circuits is considered to be the next step towards the realization of novel logic and memory devices. Here, we report on an ordered 2D network of neighboring ternary switching units represented by triazatruxene (TAT) molecules organized in a honeycomb lattice on a Ag(111) surface. Using low-temperature scanning tunneling microscopy, we are able to control the bonding configurations of individual TAT molecules within the lattice, realizing up to 12 distinct states per molecule. The switching between those states shows a particularly strong bias dependence ranging from tens of millivolts to volts. Based on a single TAT molecule as a fundamental building block, we then explore the low-bias switching behavior in units consisting of two and more interacting TAT molecules purposefully defined by the high-bias switching within the honeycomb lattice. we demonstrate the possibility to realize up to 9 and 19 distinguishable states in a dyad and a tetrad of coupled switching units, respectively. The switching dynamics can be triggered and accessed by single-point measurements on a single molecule. High experimental control over the desired state, owing to hierarchical switching and pronounced switching directionality, as well as the observed full reversibility, makes this system particularly appealing, paving the way to design complex molecule-based memory systems. This article is protected by copyright. All rights reserved.

Ferrocenyl-Substituted Triazatruxenes: Synthesis, Electronic Properties, and the Impact of Ferrocenyl Residues on Directional On-Surface Switching on Ag(111).

We report on seven new ferrocenyl-(1, 3)- and ferrocenylethynyl-modified N,N',N″-triethyltriazatruxenes (EtTATs) 4-7 as well as the dodecyl counterpart 2 of compound 1 and their use as molecular switching units when deposited on a Ag(111) surface. Such functional units may constitute a new approach to molecule-based high-density information storage and processing. Besides the five compounds 1-3, 6, and 7, where the 3-fold rotational symmetry of the triazatruxene (TAT) template is preserved, we also included 2-ethynylferrocenyl-TAT 4 and 2,2'-di(ethynylferrocenyl)-TAT 5, whose mono- and disubstitution patterns break the 3-fold symmetry of the TAT core. Voltammetric studies indicate that the ferrocenyl residues of compounds 1-7 oxidize prior to the oxidation of the TAT core. We have noted strong electrostatic effects on TAT oxidation in the 2,2',2″-triferrocenyl-TAT derivatives 1 and 2 and the 3,3',3″-isomer 3. The oxidized complexes feature multiple electronic excitations in the near-infrared and the visible spectra, which are assigned to dδ/δ* transitions of the ferrocenium (Fc+) moieties, as well as TAT → Fc+ charge-transfer transitions. The latter are augmented by intervalence charge-transfer contributions Fc → Fc+ in mixed-valent states, where only a part of the available ferrocenyl residues is oxidized. EtTAT was previously identified as a directional three-level switching unit when deposited on Ag(111) and constitutes a trinary-digit unit for on-surface information storage. The symmetrically trisubstituted compound 6 retains this property, albeit at somewhat reduced switching rates due to the additional interaction between the ferrocenyl residues and the Ag surface. In particular, the high directionality at low bias and the inversion of the preferred sense of the on-surface rocking motion with either a clockwise or counterclockwise switching sense, depending on the identity of the surface enantiomer, are preserved. Unsymmetrical substitution in mono- and diferrocenylated 4 and 5 alters the underlying ratchet potential in a manner such that a two-state switching between the two degenerate surface conformations of 4 or a pronounced suppression of switching (5) is observed.

"That was not the discussion we wanted to have": Engagement of civil society organizations with synthetic biology.

Responsible Research and Innovation calls for comprehensive public and stakeholder involvement. Its specific requirements, however, have raised criticism concerning the limitation of engagement opportunities for actors like Civil Society Organizations that do not share mainstream perspectives on technological innovations. Our article investigates the engagement of critical Civil Society Organizations in public debates and dialogues on synthetic biology and asks how they contribute to opening up respective debates. Based on three case studies, we show how Civil Society Organizations engage in and frame the debate on synthetic biology in different organizational formats. We find that Civil Society Organizations have explicitly challenged visions of a sustainable future by airing concerns about its risks and adverse impacts and engage in ontological debates about synthetic biology. Yet, we argue that different engagement formats are needed to ensure a diverse public debate on synthetic biology.

"Are You a TA Practitioner, Then?" - Identity Constructions in Post-Normal Science.

Technology assessment (TA) is a paradigmatic case for the manifold and, at times, ambiguous processes of identity formation of researchers in inter- and transdisciplinary settings. TA combines the natural, technical, and social sciences and follows the multiple missions of scientific analysis, public outreach, and policy advice. However, despite this diversity, it also constitutes a genuine community with its own discourses, conferences, and publications. To which extent "being a TA practitioner" also provides for a genuine scholarly identity is still unclear. Building on interviews with technology assessment practitioners at an academic TA institute, we ask what inter/trans/disciplinary identification patterns emerge in this field. Our analysis shows that TA practitioners adopt multiple identities, from "enthusiastic TA practitioner" to "strong interdisciplinarian" or "disciplinarian" - with distinct identity troubles inherent in all these options. We find that generational affiliation plays a vital role in identity formation. It relates to different primary orientations (towards research or advisory practices), inter/disciplinary backgrounds, contracting modes, and lengths of time spent at the TA institute. We conclude firstly, that disciplinary categories figure strongly in transdisciplinary identities; secondly, that the relation of chronos and identity warrants more substantial consideration: as time spent at a transdisciplinary institute as or as perceived options for "futuring one's identity"; thirdly, that our understanding of academic generations could profit from a more sociological conception; and, fourthly, that TA's multidisciplinary setup and threefold orientation towards science, society, and policy result in multiplying possible identities and thus making it difficult to form a stable community.

Towards understanding vaccine hesitancy and vaccination refusal in Austria.

In Austria, data on vaccine hesitancy is scarce. Available studies suggest around 1-11% of parents refuse vaccination, while many more are hesitant and consider refraining from some but not all of the recommended vaccinations. However, the key drivers for vaccine hesitancy in Austria are largely unknown. To learn more about vaccination coverage, attitude towards and knowledge around immunization as well as views on mandatory vaccination, we conducted a survey in a rural Austrian lay population including adults and children. Two paper-based questionnaires, one for adults 16 years or older and one for children aged 6-15 years, were developed, then sent to all houses of a rural community in Austria as well as handed out at the local primary and middle school, respectively. Self-reported coverage rates of children and adults were found to be low. Within the surveyed population 3% of children had never been or do not get vaccinated. More than half (57%) of the survey participants had a positive attitude towards vaccines, 21% were without reserves, 16% were found skeptical and 5% had a generally negative attitude. Knowledge about immunization in general was poor. Younger adults and people with secondary education appear to be most skeptical and negative towards vaccination. Children's attitudes were closely linked to those of their parents. The major concern around vaccination in adults was fear of side effects. In adults, 54.2% support mandatory vaccination for Health Care Workers and 20.7% are against it. 39% of adults and 37% of children wanted more information on vaccination, preferably provided by physicians. Knowledge about disease prevention by vaccination should be improved and children could also benefit from an early age-appropriate vaccine education to strengthen health literacy. Physicians are the most trusted source of health information. Medical doctors should be aware of their very important role in transmitting trusted health information. This should include an up-to-date education in communicable disease prevention and immunization during their whole medical career. Furthermore, the curricula of health-care workers may need to be improved and harmonized concerning prevention and vaccination.

Let's (not) talk about synthetic biology: Framing an emerging technology in public and stakeholder dialogues.

Synthetic biology is an emerging technoscience, which, so far, lacks a broader public debate. To foster early societal dialogue, a range of public engagement events have been initiated over the past decade. This article discusses the configurations of the emerging debate on synthetic biology in the context of the EU FP7 project SYNENERGENE. Drawing on notions of frames and framing in media studies and policy analysis, we ask which distinct frames are invoked and become dominant in current discussions about synthetic biology. Our analysis indicates significant reconfigurations in the framing of synthetic biology compared with previous biotechnology debates. Frames that traditionally served to problematize biotechnology, that is, ethics, risks, and economics, become less dominant. Instead, the potential to contribute to social progress is placed in the foreground. Moreover, discussions on ethics, risks, and governance frequently occur on an abstract level, invoking generic statements that could be made for any new technology.

Tip-Induced Inversion of the Chirality of a Molecule's Adsorption Potential Probed by the Switching Directionality.

The switching behavior of surface-supported molecular units excited by current, light, or mechanical forces is determined by the shape of the adsorption potential. The ability to tailor the energy landscape in which a molecule resides at a surface gives the possibility of imposing a desired response, which is of paramount importance for the realization of molecular electronic units. Here, by means of scanning tunneling microscopy, a triazatruxene (TAT) molecule on Ag(111) is studied, which shows a switching behavior characterized by transitions of the molecule between three states, and which is attributed to three energetically degenerate bonding configurations. Upon tunneling current injection, the system can be excited and continuously driven, showing a switching directionality close to 100%. Two surface enantiomers of TAT show opposite switching directions pointing at the chirality of the energy landscape of the adsorption potential as a key ingredient for directional switching. Further, it is shown that by tuning the tunneling parameters, the symmetry of the adsorption potential can be controllably adjusted, leading to a suppression of the directionality or an inversion of the switching direction. The findings represent a molecule-surface model system exhibiting unprecedented control of the shape of its adsorption potential.

Interaction of host cellular factor ANP32B with matrix proteins of different paramyxoviruses.

Although most non-segmented negative-strand RNA viruses (NNSVs) replicate in the cytoplasm, NNSV proteins often exert host manipulatory functions in the nucleus. Matrix (M) proteins of henipaviruses and other paramyxoviruses shuttle through the nucleus, where host factors may bind for M modification or host-cell manipulation. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) is an interactor of Hendra and Nipah virus M. Both accumulate in the nucleus in an ANP32B-dependent manner. Here we demonstrate that the nuclear localization signal (NLS) of ANP32B is dispensable for HeV M binding. Specific purification of M-ANP32B but not of M-ANP32A complexes revealed that neither the negatively charged acidic nor the leucine-rich regions of ANP32 proteins per se mediate interactions with henipavirus M proteins. Whereas pneumovirus M did not interact with ANP32B, Newcastle disease virus (NDV, genus Avulavirus), Sendai virus (SeV, genus Respirovirus), Measles virus (MeV, genus Morbillivirus) and Canine distemper virus (CDV, genus Morbillivirus) M were able to form complexes with ANP32B. However, in contrast to NDV M and SeV M, which accumulated in the nucleus ANP32B dependently, both morbillivirus Ms did not accumulate in the nucleus, neither at ANP32B overexpression nor after nuclear protein export inhibition. These results indicate that intracellular compartmentalization of cytoplasmic morbillivirus M and nuclear ANP32B prevented an intracellular interaction. Overall, we provide evidence for a general ability of paramyxovirus M proteins to interact with ANP32B. This suggests a conserved, yet to be clarified mechanism might play a role in host manipulation and immune regulation in infected hosts.

Selection of differently temporally regulated African swine fever virus promoters with variable expression activities and their application for transient and recombinant virus mediated gene expression.

African swine fever virus threatens pig production worldwide due to the lack of vaccines, for which generation of both deletion and insertion mutants is considered. For development of the latter, operational ASFV promoters of different temporal regulation and strengths are desirable. We therefore compared the capacities of putative promoter sequences from p72, CD2v, p30, viral DNA polymerase and U104L genes to mediate expression of luciferase from transfected plasmids after activation in trans, or p30-, DNA polymerase- and U104L promoters in cis, using respective ASFV recombinants. We identified sequences with promoter activities upstream the viral ORFs, and showed that they differ in both their expression intensity regulating properties and in their temporal regulation. In summary, p30 and DNA polymerase promoters are recommended for high level early regulated transgene expression. For late expression, the p72, CD2v and U104L promoter are suitable. The latter however, only if low level transgene expression is aimed.

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription.

UNLABELLED: Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCE: Regulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well.

Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

UNLABELLED: Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 µm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 µm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 µm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE: Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

Rabies Virus Hijacks and accelerates the p75NTR retrograde axonal transport machinery.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.

ANP32B is a nuclear target of henipavirus M proteins.

Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.