Monitoring of bactericidal action of laser by in vivo imaging of bioluminescent E. coli in a cutaneous wound infection

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. This study aimed to evaluate the bactericidal action of an 810-nm diode laser in a cutaneous wound infection. An Escherichia coli strain was transformed with a shuttle vector (pRB474) containing firefly luciferase gene from Photinus pyralis resulting in a bioluminescent phenotype. Because firefly luciferase is an enzyme and as such is prone to inactivation at elevated temperature, the first phase has consisted in evaluating in vitro the effect of temperature elevation (30, 40, 50, and 60°C for 2 min) on bacteria bioluminescence. The second phase was performed in vivo. Two full-thickness circular, 14-mm diameter wounds (control and laser-irradiated) were induced on rats. Wound infection was carried out using a suspension (50 μl PBS) containing 5x10⁷ cells of bioluminescent E. coli (10⁹ cells/ml). Thirty minutes later, light irradiation was performed with an 810-nm diode laser (P=10 W, ∅=1.4 cm, fluence: 130, 195, and 260 J/cm²). Temperature was measured within each wound with a noncontact infrared thermometer. Light emission of the bioluminescent bacteria was monitored in vivo by a bioluminescence imaging system before and at 4, 8, 24, and 48 h after laser irradiation. In vitro, bacteria bioluminescence is not affected when temperature is maintained at 50°C for 2 min. In vivo, bioluminescence imaging showed that at 4 h, the viability of E. coli was reduced when compared to the control (CTRL) group (p<0.01). This observation was confirmed at 8 h (p<0.001), at 24 h (p<0.001), and finally at 48 h (p<0.001). Loss of viability of E. coli depends on laser fluence. At 48 h, bioluminescent bacteria were not detected (100% loss of viability) in the wound irradiated at 260 J/cm². For this fluence, the temperature reached 45°C at the end of the irradiation. This study confirms previous observations on the bactericidal effect of diode lasers. Because a progressive desiccation of the superficial dermis is usually observed when using laser irradiation, the hypothesis that laser irradiation dries out the wound making the wound an inhospitable place for bacteria is much more relevant than a direct effect of infrared light on chromophores inside bacteria. This is confirmed by the fact that in this latter case, one would expect an immediate drop in luminescence followed by an increase as the surviving bacteria started to divide and repopulate the wound. However, the exact mechanism deserves further studies. This study points out the advantage of using bioluminescence imaging to evaluate laser for the treatment of acute infections in vivo, nondestructively, and noninvasively.     

“萤火虫”成像技术对甲状腺乳头状癌诊断的临床意义

目的对比分析＂萤火虫＂成像技术和二维高频超声显示甲状腺内细砂粒样钙化对甲状腺乳头状癌诊断的临床意义。方法选取我院甲状腺外科2010年1月～2010年7月128例甲状腺结节手术患者（共计160个结节）,良性结节96个,恶性结节64个,其中甲状腺乳头状癌结节63个,甲状腺髓样癌结节1个。术前患者均进行甲状腺二维高频超声及＂萤火虫＂成像技术检查,重点观察甲状腺结节内有无细砂粒样钙化,诊断甲状腺乳头状癌,并与术后病理结果对照分析。结果二维高频超声检出甲状腺内细砂粒样钙化诊断甲状腺乳头状癌的敏感性54.0%（34/63）、特异性78.4%（76/97）、精确度68.8%（110/160）;＂萤火虫＂成像技术诊断甲状腺癌的敏感性81.0%（51/63）、特异性81.4%（80/97）、精确度81.3%（130/160）。两者对比有显著差异性（P〈0.05）。结论 ＂萤火虫＂成像技术在检出细砂粒样钙化诊断甲状腺乳头状癌的敏感性、特异性、精确度均高于二维高频超声。     

Firefly luciferase terminally degraded by mild heat exposure: implications for reporter assays

Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and β-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with β-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.     

Kinetic characterization and in vitro toxicity evaluation of a luciferase less susceptible to HPV chemical inhibition

Enzyme-based in vitro toxicity assays are often susceptible to inhibition by test compounds. A mutant luciferase selected to be less susceptible to inhibition by chloroform (CNBLuc03-06) and other high production volume (HPV) chemicals, consisting of three point mutations was created and characterized. The mutant luciferase was less inhibited by chloroform, other HPV chemicals and common surfactant release reagents (Triton-X and SDS) compared to the wild-type. Inhibition was shown to be competitive. CNBLuc03-06 was a factor of 1.5–3.2 more active than wild type and exhibited a much higher affinity for ATP. CNBLuc03-06 was more thermostable than wild-type and also more active at pH values higher than 10. Both luciferases exhibited a significant tradeoff between activation and susceptibility to chemical inhibition in the presences of the reducing agent DTT. Inhibition to HPV chemicals was eliminated using an “optimum” formulation of DTT and co-solvent ethanol. The performance of CNBLuc03-06 in cell-based in vitro toxicity assays was shown to be superior to the current commercial formulation.     

转虫荧光素酶基因细胞在亲电性化学污染物监测中的应用研究

目的 用亲电性反应元件(EPRE)调控的转虫荧光素酶基因细胞快速评价环境的污染程度.方法 采用分子生物学的方法,将EPRE与TK基本启动子和编码虫荧光素酶(LUC)的基因相融合,构建成EPRE调控的LUC稳定表达载体,将其转染于HeLa细胞,经G418筛选出稳定的细胞株.该细胞经不同浓度的亚砷酸钠(NaAsO2)、氯化镉(CdCl2)、氯化汞(HgCl2)和顺丁烯乙酸二乙酯(DEM)作用后,用荧光素酶试剂盒物测LUC的表达量.结果 DNA测序显示,EPRE调控的LUC稳定表达载体结构框架正确;LUC的表达量与受试物具有一定的剂量-效应关系,其中DEM的剂量-效应关系最明显.结论 EPRE调控的转LUC基因细胞构建成功.     

Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140

Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC₅₀ values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI₅₀ values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9–Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.     

萤火虫荧光素酶报告基因重组逆转录病毒载体的构建及鉴定

目的构建含萤火虫荧光素酶报告基因的逆转录病毒载体,为进一步研究生物发光活体动物体内光学成像奠定基础。方法利用重组DNA技术,将荧光素酶报告基因载体pGL3-Basic双酶切得到的目的基因fluc+亚克隆至逆转录病毒载体pLXSN中,重组质粒pL(fluc)SN在脂质体介导下乒乓转染GP+E86和PA317包装细胞,G418筛选,直至出现抗性克隆。扩大培养,测定病毒滴度,并检测荧光素酶活性。结果经限制性酶切分析鉴定,载体插入基因大小、位置均正确,并用GP+E86和PA317细胞进行包装、病毒滴度测定、筛选,建立具有较高滴度的重组逆转录病毒fluc细胞系,该细胞可高效表达荧光素酶。结论成功构建了重组质粒pL(fluc)SN,为标识干细胞进行基础研究提供了一种检测方法和平台。     

Non-invasive visualisation of the development of peritoneal carcinomatosis and tumour regression after <Superscript>213</Superscript>Bi-radioimmunotherapy using bioluminescence imaging

Purpose Non-invasive imaging of tumour development remains a challenge, especially for tumours in the intraperitoneal cavity. Therefore, the aim of this study was the visualisation of both the development of peritoneal carcinomatosis and tumour regression after radioimmunotherapy with tumour-specific ²¹³Bi-Immunoconjugates, via in vivo bioluminescence imaging of firefly luciferase-transfected cells. Methods Human diffuse-type gastric cancer cells expressing mutant d9-E-cadherin were stably transfected with firefly luciferase (HSC45-M2-luc). For bioluminescence imaging, nude mice were inoculated intraperitoneally with 1 × 10⁷ HSC45-M2-luc cells. On days 4 and 8 after tumour cell inoculation, imaging was performed following D-luciferin injection using a cooled CCD camera with an image intensifier unit. For therapy, mice were injected with 2.7 MBq ²¹³Bi-d9MAb targeting d9-E-cadherin on day 8 after tumour cell inoculation. Bioluminescence images were taken every 4 days to monitor tumour development. Results After i.p. inoculation of HSC45-M2-luc cells into nude mice, development as well as localisation of peritoneal carcinomatosis could be visualised using bioluminescence imaging. Following ²¹³Bi-d9MAb therapy on day 8 after intraperitoneal inoculation of HSC45-M2-luc cells, small tumour nodules were totally eliminated and larger nodules showed a clear reduction in size on day 12 after tumour cell inoculation. Subsequently a recurrence of tumour mass was observed, starting from the remaining tumour spots. By measuring the mean grey level intensity, tumour development over time could be demonstrated. Conclusion Non-invasive bioluminescence imaging permits visualisation of the development of peritoneal carcinomatosis, localisation of tumour in the intraperitoneal cavity and evaluation of therapeutic success after ²¹³Bi-d9MAb treatment.