In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

Virus‐Inspired Polymer for Efficient In Vitro and In Vivo Gene Delivery

Clinical translation of nucleic acids drugs has been stunted by limited delivery options. Herein, we report a synthetic polymer designed to mimic viral mechanisms of delivery called VIPER (virus‐inspired polymer for endosomal release). VIPER is composed of a polycation block for condensation of nucleic acids, and a pH‐sensitive block for acid‐triggered display of a lytic peptide to promote trafficking to the cell cytosol. VIPER shows superior efficiencies compared to commercial agents when delivering genes to multiple immortalized cell lines. Importantly, in murine models, VIPER facilitates effective gene transfer to solid tumors.     

A Comparison of In‐Vitro and In‐Vivo Degradation of Poly(D,L‐lactide) Bio‐Absorbable Intra‐Medullary Plugs

Poly(D ,L ‐lactide) has been evaluated as a material for the manufacture of intra‐medullary plugs to be used in total hip arthoplasty. Plugs were manufactured by compression moulding and subjected to in‐vitro and in‐vivo degradation. In‐vitro hydrolysis was carried out by immersion in phosphate buffered saline (Ringer's solution) at 37°C and rates of degradation were relatively rapid with molecular weight halving after 30 days. In‐vivo degradation was assessed by implantation into dogs followed by retrieval at intervals up to 24 months. Molecular weight was found to reduce to half the original value in about 190 days. It is thought that this difference in degradation rate is because of diffusional control of the overall process. Histology showed that the implanted plugs were resorbed over 24 months.     

Precise In Vivo Inflammation Imaging Using In Situ Responsive Cross‐linking of Glutathione‐Modified Ultra‐Small NIR‐II Lanthanide Nanoparticles

To improve the bioimaging signal‐to‐noise ratio (SNR), long‐term imaging capability, and decrease the potential biotoxicity, an in vivo cross‐linking strategy was developed by using sub‐10 nm, glutathione‐modified, lanthanide nanoprobes. After administration, the nanoprobes cross‐link in response to reactive oxygen species (ROS) at the inflamed area and enable the quick imaging of ROS in the second near‐infrared (NIR‐II) window. These nanoprobes could be rapidly excreted due to their ultra‐small size. This strategy may also be applied to other ultra‐small contrast agents for the precise bioimaging by in situ lesion cross‐linking.     

In‐Transit Electroextraction of Small‐Molecule Pharmaceuticals from Blood

An electrokinetic platform was developed for extracting small‐molecule pharmaceuticals from a dried blood spot. Through the exclusion of liquid reagents and use of low field strength (6 V cm^?1), the electroextraction of a drug from a dried blood spot, deposited on a polymer inclusion membrane (PIM), could be realised while in transit in the mail. In transit sample preparation provides a potential solution to in situ sample degradation and may accelerate the workflow upon arrival of a patient sample at the analytical facility. The electroextraction method was enabled through our discovery of the use of 15–20 μm thin PIMs as electrophoretic separation medium in absence of liquid reagents. Here, a PIM consisting of cellulose triacetate as polymer base, 2‐nitrophenyl octyl ether as plasticizer and 1‐ethyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide as carrier was used. The PIM, was packaged with two 12 V batteries to supply the separation voltage. A blood spot containing berberine chloride was deposited and dried before the applying the separation potential, allowing for the electroextraction while the packaged device was shipped in internal mail. Upon arrival in the analytical laboratory, the PIM was analysed using a fluorescence microscope with photon multiplier tube, quantifying the berberine extracted away from the sample matrix. This platform represents a new opportunity for processing clinical samples during transport to the laboratory, saving time and manual handling to accelerate the time to result.