Originpro 2019b

Reconstructed neurons were merged and aligned the center of their soma. The combined reconstructed neurons were placed in a Cartesian grid and the average length of each neuronal segment within grid-boxes of 25 × 25 µm was calculated, yielding a raw density matrix. To obtain the axo-dendritic overlap, the axonal matrix was multiplied by the dendritic matrix and then normalized to the sum of all values. The matrices were then transferred to OriginPro 2019b (Origin Lab) to create a contour plot.

An osmotic fragility test was performed on freshly prepared RBCs (untreated and treated with β-Crt) following a classical procedure [25 (link)] with some modifications. Each sample (i–v) was equally divided and added to a series of 30 hypotonic solutions with a NaCl content ranging from 0.9% to 0%, incubated for 10 min at 4 °C, and centrifuged as described above. Absorption spectra of the collected supernatants were recorded in the range of 280–700 nm on a Cary 50 Bio-spectrophotometer (Varian). The haemolysis rate was determined as described previously [26 (link)]. The spectra in the range of 460–700 nm were analysed with OriginPro 2019b (OriginLab) using a combination of exponential and Gaussian functions to calculate the area under the spectrum, with the maximum at 577 nm (indicator of the amount of released Hb). The normalised haemolysis curves, as a function of NaCl concentration, are of a sigmoidal character. They were fitted using a basic Boltzmann function.

The data were organized using Microsoft Excel software, analyzed by ANOVA using SPSS 17.0 statistical software, and the least significant difference (LSD) method was used to compare the differences between groups of different data, with the level of significance set at α = 0.05. Graphing was performed using Origin Pro 2019b (Origin Lab Corporation, Inc. Northampton, Massachusetts, USA) software for graphing.

All strains were cultured overnight on MHA plates (Biomaxima, Lublin, Poland) at 37 °C and suspended in saline (0.9% NaCl, Chempur) to OD₆₀₀ = 0.1 (spectrophotometer HACH LANGE GmbH, Berlin, Germany). Two media were selected for this experiment: minimal MOPS medium and complex LB medium. The media pH was set in a range between 5.0 and 8.0 with 0.5 steps using 36 % HCl and 10 M NaOH solutions (Mettler Toledo Quattro pH meter, MP220 Basic, Columbus, OH, USA). Further, 100 µL of the medium at a specific pH was added to the well and inoculated with 10 µL of bacterial solutions to a final density of 1 × 10⁶ CFU/mL. All experiments were performed in triplicate on three different days. The plates we incubated with agitation for 24 h at 37 °C and the absorbances (OD₆₀₀) were measured (VarioSkan LUX, Thermo Fisher Scientific, Waltham, MA, USA). The statistical analysis was performed using one-way ANOVA and the Levene test, followed by the Tukey test in the OriginPro 2019b (OriginLab Corporation, Northampton, USA) software (p < 0.05).

Data analysis using the two-tailed t-test, Kolmogorov-Smirnov normal distribution analysis, Grubbs’s test for the identification of outliers, and correlation studies (linear fit with calculation of Pearson’s correlation coefficient) was carried out using OriginPro 2019b (OriginLab Corporation, Northampton, MA, United States).

For a total volume of 100 µl, CTB–LPETGA (200 µM) and appropriate peptide/glycopeptide (3 mM, 15 equiv) were mixed, and the reaction mixture was made up to volume with the HEPES buffer {+DMSO [10% v/v (final)], if required} before the addition of Sortase 7M (30 µM, 15 mol%). The reaction mixture was incubated at 25°C, and timepoints for analysis by SDS-PAGE were taken at 2, 5, 10, 20, 30, 60, 90, and 120 min. For time point samples, 5 µl of the reaction was diluted in 15 µl H₂O and mixed with 5 × SDS loading buffer (5 µl). Samples were heated to 95°C for 5 min before being frozen and stored at −20°C before analysis by SDS-PAGE. Samples were run on a 15% Tris-glycine SDS-PAGE gel, stained with Coomassie blue (12 h), destained, and imaged using a Bio-Rad Gel Doc XR system. The relative quantity of labeled product and unlabeled starting material was quantified by densitometry (Bio-Rad Image Lab 6.0.1, Bio-Rad Laboratories Ltd.). Data were analyzed in OriginPro 2019b (OriginLab Corporation, Northampton, MA, United States). Original SDS-PAGE gels are included within the Supplementary Material.