A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes

地球外有機化合物に対する微生物代謝の解明から全く新規な酵素系を発見 --生命分子進化の理解や産業応用に期待--. 京都大学プレスリリース. 2021-01-20.The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C–H bond in methyl groups need to be activated

Randomised, multicentre prospective trial of transarterial chemoembolisation (TACE) plus sorafenib as compared with TACE alone in patients with hepatocellular carcinoma: TACTICS trial.

OBJECTIVE:This trial compared the efficacy and safety of transarterial chemoembolisation (TACE) plus sorafenib with TACE alone using a newly established TACE-specific endpoint and pre-treatment of sorafenib before initial TACE. DESIGN:Patients with unresectable hepatocellular carcinoma (HCC) were randomised to TACE plus sorafenib (n=80) or TACE alone (n=76). Patients in the combination group received sorafenib 400 mg once daily for 2-3 weeks before TACE, followed by 800 mg once daily during on-demand conventional TACE sessions until time to untreatable (unTACEable) progression (TTUP), defined as untreatable tumour progression, transient deterioration to Child-Pugh C or appearance of vascular invasion/extrahepatic spread. Co-primary endpoints were progression-free survival (PFS), which is not a conventional one but defined as TTUP, or time to any cause of death plus overall survival (OS). Multiplicity was adjusted by gatekeeping hierarchical testing. RESULTS:Median PFS was significantly longer in the TACE plus sorafenib than in the TACE alone group (25.2 vs 13.5 months; p=0.006). OS was not analysed because only 73.6% of OS events were reached. Median TTUP (26.7 vs 20.6 months; p=0.02) was also significantly longer in the TACE plus sorafenib group. OS at 1 year and 2 years in TACE plus sorafenib group and TACE alone group were 96.2% and 82.7% and 77.2% and 64.6%, respectively. There were no unexpected toxicities. CONCLUSION:TACE plus sorafenib significantly improved PFS over TACE alone in patients with unresectable HCC. Adverse events were consistent with those of previous TACE combination trials. TRIAL REGISTRATION NUMBER:NCT01217034

Final Results of TACTICS: A Randomized, Prospective Trial Comparing Transarterial Chemoembolization Plus Sorafenib to Transarteria Chemoembolization Alone in Patients with Unresectable Hepatocellular Carcinoma

IntroductionSeveral clinical trials comparing the efficacy and safety of transarterial chemoembolization (TACE) plus molecular-targeted agents versus TACE alone revealed no clinical benefits in progression-free survival (PFS) or overall survival (OS). Here, we report the final OS analysis from the TACTICS trial, which previously demonstrated significant improvement in PFS with TACE plus sorafenib in patients with unresectable hepatocellular carcinoma (HCC) (NCT01217034).MethodsPatients with unresectable HCC were randomized to a TACE plus sorafenib group (N = 80) or a TACE alone group (N = 76). Patients in the combination treatment group received sorafenib 400 mg once daily for 2-3 weeks before TACE, followed by 800 mg once daily during on-demand conventional TACE sessions until time to untreatable progression. In this trial, TACE-specific PFS was used. TACE-specific PFS is defined as the time from randomization to progressive disease (PD) or death from any cause, and PD was defined as untreatable progression, caused by the inability of a patient to further receive or benefit from TACE for reasons that include intrahepatic tumor progression (25% increase vs. baseline) according to response evaluation criteria in cancer of the liver, the detection of extrahepatic spread, vascular invasion, or transient deterioration of liver function to Child-Pugh C after TACE.ResultsAt the cut-off date of July 31, 2020, 131 OS events were observed. The median OS was 36.2 months with TACE plus sorafenib and 30.8 months with TACE alone (hazard ratio [HR] = 0.861; 95% confidence interval [CI], 0.607-1.223; p = 0.40, ΔOS, 5.4 months). The updated PFS was 22.8 months with TACE plus sorafenib and 13.5 months with TACE alone (HR = 0.661; 95% CI, 0.466-0.938; p = 0.02). Post-trial treatments with active procedures/agents were received by 47 (58.8%) patients in the TACE plus sorafenib group and 58 (76.3%) in the TACE alone group (p = 0.01). In post hoc analysis, PFS and OS benefit were shown in HCC patients with tumor burden beyond up-to-7 criteria.ConclusionsIn TACTICS trial, TACE plus sorafenib did not show significant OS benefit over TACE alone; however, clinical meaningful OS prolongation and significantly improved PFS was observed. Thus, the TACE plus sorafenib can be considered a choice of treatment in intermediate-stage HCC, especially in patients with high tumor burden. Trial Registration: NCT01217034

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma

Characterization of hepatitis B virus with complex structural variations

Abstract Background Hepatitis B virus (HBV) infection is one of the most serious public health issues. Recent HBV genetic research has revealed novel genetic rearrangements termed complex structural variations (SVs), which are composed of combinations of SVs such as insertions, deletions, and duplications. An extensive search was made for complex SVs of HBV and their characteristics were analyzed. Results Fifty-five HBV strains with complex SVs were identified by analyzing genetic sequences of HBV with bioinformatical tools. Along with 15 HBV strains with complex SVs in a previous report, a total of 70 HBV strains harboring complex SVs were analyzed. Complex SVs in the HBV genome were located frequently between nt 1500 and 2000. Insertions were observed in 65/70 (92.9%) of HBV strains with complex SVs. As insertional motif sequences, hepatocyte nuclear factor 1 binding site, a sequence complementary to part of box α in enhancer II, and insertions of unknown origins were observed. The complex SVs were classified into six groups, and combination of insertion and deletion was observed more frequently than other patterns. Conclusion Through an extensive search of HBV sequences, new strains with complex SVs were identified in this study. Characteristics of HBV with complex SVs were clarified by the analysis of 70 HBV strains harboring complex SVs. Further investigation is required to elucidate its role in pathogenesis of HBV-related liver disease