Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing.

The mismatch of HLA haplotypes between donor and recipient adversely affects the outcome of tissue transplantation. TheB2Mgene knockout (B2M-KO) disrupts the HLA I heterodimer formation; therefore,B2M-KO cells have reduced immunogenicity to allogeneic CD8+ T cells. Thus, theB2M-KO IPSCs and their derivatives can potentially solve a problem of the immunological compatibility in allogeneic transplantations. Using CRISPR/Cas9-mediated genome editing, we generated a human B2M-KO iPSC line (RCPCMi007-A-1). The RCPCMi007-A-1 iPSCs express pluripotency markers, have typical stem cell morphology, maintain normal karyotype, and the ability to differentiate into three germ layers.

Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells.

The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.

Medicinal leech antimicrobial peptides lacking toxicity represent a promising alternative strategy to combat antibiotic-resistant pathogens.

The rise of antibiotic resistance has necessitated the development of alternative strategies for the treatment of infectious diseases. Antimicrobial peptides (AMPs), components of the innate immune response in various organisms, are promising next-generation drugs against bacterial infections. The ability of the medicinal leech Hirudo medicinalis to store blood for months with little change has attracted interest regarding the identification of novel AMPs in this organism. In this study, we employed computational algorithms to the medicinal leech genome assembly to identify amino acid sequences encoding potential AMPs. Then, we synthesized twelve candidate AMPs identified by the algorithms, determined their secondary structures, measured minimal inhibitory concentrations against three bacterial species (Escherichia coli, Bacillus subtilis, and Chlamydia thrachomatis), and assayed cytotoxic and haemolytic activities. Eight of twelve candidate AMPs possessed antimicrobial activity, and only two of them, 3967 (FRIMRILRVLKL) and 536-1 (RWRLVCFLCRRKKV), exhibited inhibition of growth of all tested bacterial species at a minimal inhibitory concentration of 10 µmol. Thus, we evidence the utility of the developed computational algorithms for the identification of AMPs with low toxicity and haemolytic activity in the medicinal leech genome assembly.

Role of isopeptidolysis in the process of thrombolysis.

INTRODUCTION: Known thrombolytic agents either break peptide bonds in the fibrin molecule or act as plasminogen activators, which also results in peptide bond cleavage. In thrombi, fibrin molecules are known to be cross-linked by isopeptide bonds, the formation of which is mediated by factor XIIIa. In this work, we studied the dissolution of thrombi via isopeptide bond cleavage using a recombinant destabilase. Destabilase is an enzyme secreted from the medicinal leech salivary gland. This enzyme exhibits muramidase (lysozyme) activity, in addition to endo-ε-(γ-Glu)-Lys-isopeptidase activity, which is responsible for isopeptide bond cleavage. METHODS: Venous (jugular vein) and arterial (carotid artery) thrombosis was induced in rats. Rats were intravenously injected with both recombinant destabilase produced in Escherichia coli and a commercial streptokinase preparation. After 24 h, the weight and degree of cross-linking in the thrombi were analysed. Amidolytic activity in rat blood serum was measured in order to evaluate destabilase levels in the blood. RESULTS: Destabilase was definitively shown to cause a 47.6% and 74.6% decrease in the weight of venous and arterial thrombi, respectively. The enzyme proved to be more efficient at dissolving thrombi compared to streptokinase. The combined administration of destabilase and streptokinase has a greater effect than the injection of individual enzymes. Destabilase reduces fibrin stabilization in thrombi. CONCLUSION: Cumulatively, we find that the medicinal leech destabilase is a more efficient thrombolytic agent for dissolving thrombi, which could help increase the overall effectiveness of conventional thrombolytic drugs.