Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells

Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

Pharmacological correction of morphofunctional retinal injury using 11-amino acid fragment of darbepoetin in the experiment

Introduction: The retinoprotective effect of the 11-amino acid fragment of darbepoetin PRK-002 on the models of hypertensive retinal angiopathy and hypertensive neuroretinopathy in Wistar rats was investigated in comparison with carbamylated darbepoetin and sulodexide. Materials and methods: The protective effects of the pharmacological agents were assessed using the following criteria: a semi-quantitative assessment of changes in the eye fundus when performing ophthalmoscopy, the retinal blood flow, the b/a coefficient, eNOs expression in retinal vessels, specific number of neuronal nuclei in the inner nuclear layer, and p53 expression in the retina. Results and discussion: A pronounced protective effect, exceeding sulodexide at a dose of 150 LRU/kg and carbamylated darbepoetin at a dose of 300 μg/kg when correcting retinal angiopathy was observed in PRK-002 at a dose of 4 µg/kg, which expressed in adjustment of the retinal vessels’ calibers, removing retinal arterio-venous crossings, reaching the target levels of the retinal microcirculation, the b/a coefficient, and the restoration of eNOs expression in the endothelium of retinal vessels. PRK-002 at a dose of 4 µg/kg has a pronounced neuroprotective effect comparable to carbamylated darbepoetin at a dose of 300 µg/kg in correction of hypertensive neuroretinopathy, which expressed in the normalization of the fundus image, reaching the b/a target values, the specific number of neuronal nuclei in the inner nuclear layer, inhibition of p53 expression in the neurons of the inner nuclear and ganglionic layers. Conclusion: The study revealed angio- and neuroprotective activity of the 11-amino acid fragment of darbepoetin PRK-002 in correction of retinal injury formed on the background of hypertension.

Synthesis of monomers for the preparation of optically active poly(amido–imide)s. Part 1. Synthesis of chiral imides containing a fragment of the natural amino acid based on nitrophenylcycloalkanedicarboxylic acids

Previously it was shown that the stereochemical result of the alkylation reaction of benzene with cycloalkanedicarboxylic acids depends on the order of mixing the reagents. The resulting diastereomerically pure derivatives can be used as precursors of monomers for the synthesis of optically active poly(amido–imide)s, which are basic materials in chiral chromatographic separation. Their potential for use in chiral catalytic systems, liquid crystals in ferroelectric and nonlinear optics, in the manufacture of electrodes for enantioselective recognition during bioelectrosynthesis, membrane separation technology, etc. is shown. Nitration reactions of alkyl derivatives were carried out with their subsequent imidization with natural amino acids. The resulting diastereomerically pure dicarboxylic acids are nitrated with low selectivity, unlike their anhydrides, so the nitro derivatives were synthesized by nitrating the anhydrides with anhydrous nitric acid in chloroform solution. The racemization of the α-carbon center of the amino acid fragment occurs during the imidization reaction in glacial acetic acid. Using of DMF as a solvent under mild conditions is preferable, as it eliminates the possibility of racemization of the chiral center of the amino acid fragment. It was also found that during the preparation of imides, the configuration of the chiral centers of the cycloalkane dicarboxylic acid fragment is preserved. Nitrophenylnorbornanedicarboxylic acid forms an anhydride directly during synthesis in acetic acid, unlike cyclohexanedicarboxylic acids, due to its spatial structure. Nitrophenylnorbornanedicarboxylic acid is also characterized by easier imidization. The structure was determined using 1H, 13C NMR, 1H-1H NOESY, 1H-1H COSY, HPLC, capillary electrophoresis.

Chiral β3-isocyanopropionates for multicomponent synthesis of peptides and depsipeptides containing a β-amino acid fragment

Chiral β3-isocyanopropionic acids derivatives is a new type of isocyanides for multicomponent reactions. The use of these isocyanides in Ugi and Passerini reactions allows to prepare short peptides and depsipeptides with β-amino acid moiety in the structure.

Manganism and Parkinson's disease: Mn(ii) and Zn(ii) interaction with a 30-amino acid fragment

A 30-amino acid fragment, Ac-SPDEKHELMIQLQKLDYTVGFCGDGANDCG-Am, from residues 1164 to 1193 in the encoded protein from Parkinson's disease gene Park9 (YPk9), was studied for manganese and zinc binding.