Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells—i.e., host translation—to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections.

Background: Patients in Japan with hepatocellular carcinoma (HCC) who are indicated for active treatment have a prolonged, healthy life expectancy. This study aimed to determine the effects and toxicity of transcatheter arterial chemoembolization (TACE) using cis-diamminedichloroplatinum (II) (CDDP) without Lipiodol in super-elderly patients with HCC.Methods: Transcatheter arterial chemoembolization proceeded in patients aged > 85 years with HCC as follows: hepatocellular carcinoma was evaluated using angiography, CT-portography, and CT. Then the treatment area was decided. Powdered CDDP (100 mg in 70 mL of saline) was injected into the optimal hepatic artery using a syringe pump at a rate of 3 e 4 mL/min. Embolic materials were administered until hemostasis was achieved. Details of TACE, survival rates, overall response rates (m-RECIST criteria), and toxicity (CTCAE ver. 4.0) were evaluated.Results: We retrospectively evaluated 23 procedures in 13 patients (male, n ¼ 9, female, n ¼ 4; median age, 85 (85 e 89) years) between January 2014 and December 2019. The numbers of patients with Child-Pugh classes A, B and C were 23, 0 and 0, respectively, and 3, 4, 3 and 3 patients had HBV, HCV, HBV+HCV, and NBNC, respectively. The median age at the time of treatment was 86 years and the patients underwent a median of two procedures. The CDDP dose was 58.3 mg/m 2 (recommended dose, 65 mg/m 2 ) and the embolic materials comprised gelatin sponge particles in 22 procedures and HepaSpheres in one. The areas treated during the procedures comprised the whole liver (n ¼ 13), lobes (n ¼ 2), segments (n ¼ 6), and sub-segments (n ¼ 2). The mean follow-up period was 820 days and the survival rates at 0.5, 1 and 2 years were 84.6%, 67.3%, 56.1%, respectively. The overall response and disease control rates were 78.3% and 87.0%, respectively, without severe adverse events. The median hospital stay was 6 (5 e 9 days). Conclusion:Transcatheter arterial chemoembolization using CDDP without Lipiodol is safe and effective for super-elderly patients with HCC.