N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum.

Frequency gradients during two different forms of fibrillation in the canine atria.

BACKGROUND: Atrial fibrillation (AF) is thought to be sustained by multiple reentrant wavelets or firing foci. OBJECTIVE: The aim of this study was to compare the spectral domain characteristics in the left atrium (LA) and right atrium (RA) in two different models of AF. METHODS: Rectangular 8 x 14 electrode arrays were placed on the LA and RA of 14 anesthetized dogs. AF episodes were induced with burst pacing and aconitine in each dog. For each model, AF was induced from the RA in six dogs and from the LA in six dogs. Dominant frequencies (DFs) were obtained using the fast Fourier transform of the unipolar recordings obtained from each electrode of the array. Standard deviation (SD) was used to compute the frequency dispersion within an atrium. Regularity of the signal was quantified using an organization index (OI). RESULTS: DFs were largest in the atrium where aconitine was applied. Aconitine AF had larger gradients than burst-pacing AF (5.0 +/- 4.5 vs. 0.9 +/- 1.0 Hz: P <.006). Aconitine AF when compared with burst-pacing AF had greater absolute LA-RA differences in the SD of DFs (2.3 +/- 1.9 vs. 0.2 +/- 0.2 Hz; P <.001) and in OI (0.11 +/- 0.07 vs. 0.06 +/- 0.07; P <.07). CONCLUSIONS: Differences in frequency gradients and organization were observed during AF induced by burst pacing and aconitine. This suggests that different mechanisms of AF are possible and may be identified with frequency domain analysis.