Broadness and specificity: ArdB, ArdA, and Ocr against various restriction-modification systems.

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

New temperature-switchable acyl homoserine lactone-regulated expression vector.

Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichia coli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A. logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22 °C and stops when it rises to 37 °C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1 nM to 10 µM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein. KEY POINTS: • A. logei quorum sensing system elements were applied in new expression vectors • Expression of target gene is inducible at 22 °C and it is switched off at 37 °C • Target gene expression at 22 °C is tunable by use different AI concentrations.

Bidirectional chemotherapy in gastric cancer with peritoneal metastasis combining intravenous XELOX with intraperitoneal chemotherapy with low-dose cisplatin and Doxorubicin administered as a pressurized aerosol: an open-label, Phase-2 study (PIPAC-GA2).

Background: Peritoneal metastasis (PM) develop in more than 50 % of gastric cancer (GC) patients. Median survival without treatment is not more than 3-7 months, and 8-12 months after modern combination chemotherapy. Innovative therapeutic approaches are urgently needed. Methods: Phase-2, open label prospective clinical trial assessing safety and efficacy of bidirectional chemotherapy for treating peritoneal metastasis of gastric cancer (PMGC). Treatment protocol included initial staging laparoscopy or laparotomy, 3-4 courses of systemic chemotherapy (XELOX) followed by Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC) procedures every 6 weeks until progression of disease or death. Primary endpoints were overall survival and histological peritoneal regression grading score after rebiopsy. Results: 31 patients were included (9 men, 22 women, mean age 52 years), 24 with synchronous PM at diagnosis, 7 with metachronous PM after previous chemotherapy. Mean PCI was 13.8 (min-max 6-34). XELOX was administered in all patients and combined with 56 PIPAC procedures. Complete and partial pathological response was found in 60 % of the 15 patients eligible for tumor response assessment (4 and 5 patients, respectively). Median survival was 13 months. Conclusions: Bidirectional chemotherapy combining XELOX with PIPAC with cisplatin and doxororubicin is well tolerated, can induce objective tumor regression and is associated with a promising survival in PMGC.