BackgroundThe tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli.Methodology/Principal FindingsTo extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein.ConclusionsTo our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or in combination with other global methods, such as global transcriptional machinery engineering (gTME) for strain improvements.

High-intensity focused ultrasound (HIFU) was used to treat Morris rat hepatoma 3924A implanted in the liver. Treatment was administered with a lens-focused 4-MHz transducer that created a focused beam of 550 W/cm2 at peak intensity. One hundred twelve rats with liver tumors were divided into two groups of 56 each. Group 1 received HIFU therapy while group 2 (the control group) did not. All rats were killed immediately or 1, 3, 7, 14, 21, or 28 days after treatment. Eight rats in each group were killed at each interval for pathologic and biochemical studies. Significant inhibition of the tumor growth was seen in the HIFU-treated group, with tumor growth inhibition rates of 65.4% to 93.1% from the third to the 28th day after treatment. Ultrasound-treated tumors showed direct thermal cytotoxic necrosis and fibrosis. An additional 56 ACl rats with liver tumors were divided into four groups of 14 each. Group 1 received doxorubicin hydrochloride intraperitoneally and HIFU therapy; group 2, HIFU therapy; group 3, doxorubicin hydrochloride; and group 4 (the control group), neither HIFU nor doxorubicin hydrochloride. Significantly improved survival rates were noted in HIFU-treated animals (groups 1 and 2) compared with those of groups 3 and 4. These data suggest that HIFU may be a useful method for local treatment of hepatic tumors.

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A long-term follow-up study of 144 cases with surgically and pathologically proved small hepatocellular carcinoma (less than or equal to 5 cm) from 1967 to 1987 is reported. One hundred eight cases (75.0%) were detected by alpha-fetoprotein serosurvey and/or ultrasonography mainly in a high-risk population; 129 cases (89.6%) coexisted with cirrhosis. Resection was done in 132 cases (91.7%) with three (2.3%) operative deaths; cryosurgery, laser vaporization, and hepatic arterial chemotherapy were used in the rest. Limited resection was done in 67.4% of resections. Reresection of subclinical recurrence or solitary pulmonary metastasis was done in 21 cases. The 5-year and 10-year survival rates were 67.9% and 53.4% in the resection group but zero in the nonresection group. Survival was correlated negatively with tumor size, 5-year survival after resection was 84.6% in tumors less than or equal to 2 cm but 59.5% in tumors of 4.1 to 5 cm. The increase of resectability and reresection resulted in marked improved of 5-year survival from 43.5% in 1973 to 1977 to 63.3% in 1978 to 1982 in the entire series. No significant difference was found between survival of limited resection and lobectomy. Resection may be the modality of choice for treatment of small hepatocellular carcinomas with compensated liver function. Limited resection instead of lobectomy was the key to increased resectability and decreased operative mortality in cirrhotic livers. Reresection of subclinical recurrence was important to prolong survival further.

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