Bioluminescent Optogenetics: A Novel Experimental Therapy to Promote Axon Regeneration after Peripheral Nerve Injury

Functional recovery after peripheral nerve injury (PNI) is poor, mainly due to the slow and incomplete regeneration of injured axons. Experimental therapies that increase the excitability of the injured axons have proven remarkably successful in promoting regeneration, but their clinical applicability has been limited. Bioluminescent optogenetics (BL-OG) uses luminopsins, fusion proteins of light-generating luciferase and light-sensing ion channels that could be used to increase neuronal excitability if exposed to a suitable substrate. Excitatory luminopsins were expressed in motoneurons of transgenic mice and in wildtype mice transduced with adeno-associated viral vectors. Intraperitoneal administration of coelenterazine (CTZ), a known luciferase substrate, generated intense bioluminescence in peripheral axons. This bioluminescence increased motoneuron excitability. A single administration of CTZ immediately after sciatic nerve transection and repair markedly enhanced motor axon regeneration. Compound muscle action potentials were 3–4 times larger than controls by 4 weeks after injury. The results observed with transgenic mice were comparable to those of mice in which the luminopsin was expressed using viral vectors. Significantly more motoneurons had successfully reinnervated muscle targets four weeks after nerve injury in BL-OG treated mice than in controls. Bioluminescent optogenetics is a promising therapeutic approach to enhancing axon regeneration after PNI.     

Derivatization of Antibody Fab Fragments: A Designer Enzyme for Native Protein Modification

Bioconjugates, such as antibody–drug conjugates, have gained recent attention because of their increasing use in therapeutic and diagnostic applications. Commonly used conjugation reactions based upon chemoselective reagents exhibit a number of drawbacks: most of these reactions lack regio‐ and stereospecificity, thus resulting in loss of protein functionality due to random modifications. Enzymes provide an obvious solution to this problem, but the intrinsic (natural) substrate specificities of existing enzymes pose severe limitations to the kind of modifications that can be introduced. Here we describe the application of the novel trypsin variant trypsiligase for site‐specific modification of the C terminus of a Fab antibody fragment via a stable peptide bond. The suitability of this designed biocatalyst was demonstrated by coupling the Her2‐specific Fab to artificial functionalities of either therapeutic (PEG) or diagnostic (fluorescein) relevance. In both cases we obtained homogeneously modified Fab products bearing the artificial functionality exclusively at the desired position.     

ACE2, the Receptor that Enables Infection by SARS-CoV-2: Biochemistry,Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.     

Influence of the Temperature on Coccolith-Containing Systems from Emiliania huxleyi Cultivations

Thermogravimetric analysis of a coccolith-containing biogenic broth showed a three-step degradation process. According to this system behavior, the biogenic broth was heated to specific temperatures and characterized in terms of its morphology, surface chemistry, and crystallinity. The elemental and organic composition of the treated samples was also evaluated and compared to the reference material. The presented results were acquired in an effort to exploit pretreatment scenarios for such a biogenic system that would improve and support a separation process.     

Mechanical and erosion properties of CaCO3‐EMAA thermal sprayed coatings

Polymer‐ceramic composite coatings manufactured from calcium carbonate and ethylene‐methacrylic acid copolymer (EMAA) were prepared via a thermal spray process employing different CaCO₃ filler sizes (average size of 2.8, 9 or 36 μm) and loading levels from about 2.5 to 7 wt%. The optimum filler feeding characteristics, deposition efficiency and deposition rate were obtained with a 36 μm sized CaCO₃. Tensile properties, peel strength, and the erosion resistance of a pure EMAA and CaCO₃‐EMAA composite coatings were investigated. It was found that the tensile strain at fracture of the composite coating decreased with the addition of filler to a greater degree than that observed in compression‐molded polymer composites. This is attributed to an inhomogenous distribution of the filler, with more being concentrated at the boundaries of the deposited polymer particles, thereby establishing a rigid framework within the coating. Only a small filler content is necessary to establish large changes in the mechanical properties of the coating. The peel strength of a composite coating decreases with filler content, both on a mild steel substrate and a previously sprayed polymer coating. Bonding to the latter is significantly higher and offers a possibility as a bonding layer between substrates and composite coatings. The coefficient of friction is lowered with the addition of a filler. Erosion testing has shown that the erosion resistance of PF111 is little improved overall with filler addition, although some increase is found for filler contents less than 5 vol%. Polym. Eng. Sci. 44:1448–1459, 2004. © 2004 Society of Plastics Engineers.     

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye‐Sensitized TiO2

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin ? cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on Ru^II‐dye‐sensitized TiO₂ nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria.     

Encapsulation of Hydroxyapatite Microspheres with Fluorapatite Using a Diffusion Process

Modification of hydroxyapatite surfaces can improve the properties of biomedical devices. The objective of this work was to encapsulate hydroxyapatite particles with a fluorapatite layer. A suspension of hydroxyapatite microspheres was prepared in a solution at different pH and treated with ammonium fluoride. pH, calcium, and fluoride were monitored in real time and particles subjected to fluoride analysis. After addition of fluoride, it was found that more material is released from the particle surface at low pH conditions, but leads to a high fluoride uptake from solution. Low solution fluoride levels produce a fluorapatite layer, but higher fluoride levels produce calcium fluoride.     

Technologies for Single-Cell Isolation

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.     

Glass with hydration-induced compressive stress profiles

A novel potassium phospho-aluminosilicate composition is described that can be strengthened by water vapor to achieve deep compressive stress (CS) profiles. Water vapor treatment at (A) 85°C and 85% relative humidity for 40 days results in a CS of 389 ± 20 MPa and a compressive depth of layer (DOL) of 18 ± 2 μm. When treated at (B) 160°C and 0.1 MPa for 7 days, a CS of 245 ± 20 MPa and a DOL of 40 ± 2 μm is achieved. Glasses with hydration-induced stress profiles can provide high retained strength following flaw introduction compared with ion-exchanged soda-lime silicate glass. Sample treatment B also has an exemplary Vickers indentation cracking threshold value greater than 20 kgf. The hydration profile determined by secondary ion mass spectrometry (SIMS) is shown to closely match the stress profile for these samples. SIMS analysis also shows that the depth of water enrichment correlates well with the depletion depth of phosphorus. The high tendency towards water-induced strengthening for this new type of glass even enables self-strengthening by the generation of a near-surface CS profile following exposure to ambient conditions.