Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.     

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.     

Isthmus ablation with a novel microwave catheter in dogs.

Typical atrial flutter is a macro-reentrant arrhythmia within the right atrium. Its zone of slow conduction is the so-called right atrial isthmus, representing the area between inferior vena cava, tricuspid annulus, and coronary sinus. Conduction block of this target area can terminate the arrhythmia and prevent reinduction. Isthmus ablation with radiofrequency (RF) energy is a well-established technique to produce conduction block. However, in most cases multiple energy applications are needed to create linear lesions. In individual cases, this results in prolonged procedure duration and, as a possible result of nontransmural lesions, recurrence of the arrhythmia. This article evaluates the feasibility and safety of a new steerable temperature-monitoring microwave ablation catheter in the production of linear lesions in the right atrial isthmus and examines both the dose-response relationship and the energy required for transmural lesions. Ablation was performed in eight open-chest mongrel dogs using a 2.450-MHz microwave generator with 35-50 W and a newly designed AFx 9-F deflectable catheter with a 25-mm antenna on the tip. The operation was performed under general anesthesia using nembutal and supported with room-air artificial respiration throughout the procedure. The study showed that the new deflectable AFx microwave catheter is a feasible and safe tool for creating long transmural linear lesions in the right isthmus. Based on this data, an appropriate catheter position, energy settings between 40 and 50 W, and application duration of approximately 90 s are needed. Also, the development of different antennas and curve shapes is recommended to simplify the ablation procedure and to target more complex arrhythmia substrates with microwave energy. Further studies are needed to determine the optimal anticoagulation procedure after microwave ablation.     

Bio‐orthogonal Fluorescent Labelling of Biopolymers through Inverse‐Electron‐Demand Diels–Alder Reactions

Bio‐orthogonal labelling schemes based on inverse‐electron‐demand Diels–Alder (IEDDA) cycloaddition have attracted much attention in chemical biology recently. The appealing features of this reaction, such as the fast reaction kinetics, fully bio‐orthogonal nature and high selectivity, have helped chemical biologists gain deeper understanding of biochemical processes at the molecular level. Listing the components and discussing the possibilities and limitations of these reagents, we provide a recent snapshot of the field of IEDDA‐based biomolecular manipulation with special focus on fluorescent modulation approaches through the use of bio‐orthogonalized building blocks. At the end, we discuss challenges that need to be addressed for further developments in order to overcome recent limitations and to enable researchers to answer biomolecular questions in more detail.     

The effect of milling time on the sintering kinetics of Si3N4 based nanocomposites

Multi-walled carbon nanotube (MWCNT) reinforced silicon nitride composites have been prepared by hot isostatic pressing at 20 MPa and gas pressure sintering at 2 MPa. To assure a good dispersion of the MWCNTs a highly efficient attritor milling was employed in the preparation process of the powder mixtures. The morphological and micro-structural evolution of the powder particles during the high-energy milling was monitored.We have found that the milling time has a complex influence on the structure and mechanical properties of the resulting nanocomposites through affecting both the dispersion and degradation of the nanoscale filler as well as the phase transformations of the ceramic host.     

Water vapour adsorption in highly porous carbons as seen by small and wide angle X-ray scattering

The adsorption of water vapour in a high surface area commercial carbon, as-received and after different surface chemical treatments was investigated as a function of relative humidity by three separate techniques. While gravimetry and small and wide angle X-ray scattering measurements are in reasonable agreement, volumetric observations yielded isotherms with substantially lower values for the water uptake. In each case, the results are sensitive to the surface chemistry of the sample. Several weeks were required to establish equilibrium, with strikingly different results being obtained for measurements carried out on shorter time scales. The slow kinetics of the uptake accounts for most of the discrepancies among the results of the different techniques. Immersion in water caused significant macroscopic swelling of the carbon matrix over a time scale comparable to the adsorption equilibrium time.     

Impact of Medium-Sized Extracellular Vesicles on the Transduction Efficiency of Adeno-Associated Viruses in Neuronal and Primary Astrocyte Cell Cultures

(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.     

Neuroethik – Geschichte,Definition und Gegenstandsbereich eines neuen Wissenschaftsgebiets

Definition of the problem

Fifteen years after establishing neuroethics as an international academic field, neuroethics has emerged as a vibrant, dynamic area of scientific research.

Arguments

Within a short period of a few years, specific conferences, journals, research funding programs, professional societies, and institutes were founded. Nonetheless, considerable disagreement about its definition and subject matter still remain. We argue for a differentiated conceptualization according to which the deliberate reflection of ethical problems arising from the neurosciences and their predominantly neurotechnological application belongs as much to neuroethics as does the ethical reflection of the neuroscience of morality. This does not comprise neuroscientific or neuropsychological research on morality itself, but includes the contemplation of the significance of such research for ethics and the law. Here, we provide an overview about the most important topics in neuroethics and elucidate the relevance of neuroethics for a vast diversity of societal domains reaching well beyond medicine and health care.

Conclusion

The great potential of neuroethics as a novel branch of academic research lies in finding new answers to pressing questions of high societal relevance by linking neurophilosophy and bioethics topics as well as its broad interdisciplinary network.