In the bilateral axillo-breast approach (BABA), the camera is inserted through the areolar incision, and this raises the concern it might be difficult to identify the lymph nodes (LN). The purpose of this study is to evaluate the feasibility of the Firefly for central lymph node dissection (CLND) in robotic thyroidectomy using the BABA. This study evaluated 18 patients who underwent robotic surgery using Firefly between December 2015 and March 2016. For LN mapping, 0.05 ml of ICG was injected into the thyroid 3~4 minutes before CLND. Green-stained LN could be detected easily through a near-infrared camera. The number of retrieved LNs was 7.8±3.0 after CLND using the Firefly, which was higher than the 6.7±0.2 reported in previous surgeries. In addition, it helped to distinguish between the parathyroid and the LNs. The Firefly technology was helpful in identifying the LNs, guiding the CLND and performing a complete CLND.
Fireflies* ; Humans ; Indocyanine Green ; Lymph Node Excision* ; Lymph Nodes* ; Thyroid Gland ; Thyroidectomy*

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.
Binding Sites ; Cytomegalovirus* ; Fireflies ; Humans* ; Luciferases ; Macrophages ; Permissiveness ; Transfection ; U937 Cells*

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.
Binding Sites ; Cytomegalovirus* ; Fireflies ; Humans* ; Luciferases ; Macrophages ; Permissiveness ; Transfection ; U937 Cells*

PURPOSE: Luciferase is one of the most commonly used reporter enzymes in the field of in vivo optical imaging. D-luciferin, the substrate for firefly luciferase has very high cost that allows this kind of experiment limited to small animals such as mice and rats. In this current study, we validated local injection of D-luciferin in the articular capsule for bioluminescence imaging in rabbits. MATERIALS AND METHODS: Chondrocytes were cultured and infected by replication-defective adenoviral vector encoding firefly luciferase (Fluc). Chondrocytes expressing Fluc were injected or implanted in the left knee joint. The rabbits underwent optical imaging studies after local injection of D-luciferin at 1, 5, 7, 9 days after cellular administration. We sought whether optimal imaging signals was could be by a cooled CCD camera after local injection of D-luciferin. RESULTS: Imaging signal was not observed from the left knee joint after intraperitoneal injection of D-luciferin (15 mg/kg), whereas it was observed after intraarticular injection. Photon intensity from the left knee joint of rabbits was compared between cell injected and implanted groups after intraarticular injection of D-luciferin. During the period of imaging studies, photon intensity of the cell implanted group was 5-10 times higher than that of the cell injected group. CONCLUSION: We successfully imaged chondrocytes expressing Fluc after intraarticular injection of D-luciferin. This technique may be further applied to develop new drugs for knee joint disease.
Adenoviridae ; Animals ; Chondrocytes* ; Fireflies ; Injections, Intra-Articular* ; Injections, Intraperitoneal ; Joint Capsule ; Knee Joint ; Luciferases ; Mice ; Optical Imaging ; Rabbits* ; Rats

The self-splicing group I intron from Tetrahymena thermophila has been demonstrated to perform splicing reaction with its substrate RNA in the trans configuration. In this study, we explored the potential use of the trans-splicing group I ribozymes to replace a specific RNA with a new RNA that exerts any new function we want to introduce. We have chosen thymidine phosphorylase (TP) RNA as a target RNA that is known as a valid cancer prognostic factor. Cancer-specific expression of TP RNA was first evaluated with RT-PCR analysis of RNA from patients with gastric cancer. We determined next which regions of the TP RNA are accessible to ribozymes by employing an RNA mapping strategy, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. A specific ribozyme recognizing the most accessible sequence in the TP RNA with firefly luciferase transcript as a 3' exon was then developed. The specific trans-splicing ribozyme transferred an intended 3' exon tag sequence onto the targeted TP transcripts, resulting in a more than two fold induction of the reporter activity in the presence of TP RNA in mammalian cells, compared to the absence of the target RNA. These results suggest that the Tetrahymena ribozyme can be a potent anti-cancer agent to modify TP RNAs in tumors with a new RNA harboring anti-cancer activity.
Codon, Initiator ; Exons ; Fireflies ; Humans ; Introns ; Luciferases ; RNA* ; RNA, Catalytic ; Stomach Neoplasms ; Tetrahymena ; Tetrahymena thermophila ; Thymidine Phosphorylase ; Trans-Splicing*

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Animals ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fireflies ; enzymology ; Luciferases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

PURPOSE: To demonstrate and analyze quantitatively the expression of marker genes in the Achilles tendon of rats by direct DNA injection and electroporation. MATERIALS AND METHODS: Enhanced green fluorescent protein (EGFP) cDNA or luciferase cDNA were injected into the mid-substance of rat Achilles tendon followed by various conditions of electroporation. Adenovirus encoding firefly luciferase was used to compare the transfection efficiency. Tendons were examined by confocal microscopy when EGEP was used and with luciferase, and the luminescence was measured quantitatively. RESULTS: Combined injection with electroporation increased the fluorescence intensity. The results of luciferase cDNA injection and electroporation with 25 volts/0.25 cm and 50 volts/0.25 cm showed a ten times and thirty-three times higher luminescence respectively than without using electroporation. After DNA injection and electroporation using 50 volts, the luciferase activity was slightly lower than with viral transduciton using 106 plaque forming units. CONCLUSION: Direct administration of therapeutic genes by electroporation accelerates the repair processes in musculoskeletal tissues.
Achilles Tendon* ; Adenoviridae ; Animals ; DNA ; DNA, Complementary ; Electroporation* ; Fireflies ; Fluorescence ; Gene Expression ; Luciferases ; Luminescence ; Microscopy, Confocal ; Rats* ; Tendons ; Transfection

OBJECTIVE: With the growing interests of bacteria as a targeting vector for cancer treatment, diverse genetically engineered Salmonella has been reported to be capable of targeting primary or metastatic tumor regions after intravenous injection into mouse tumor models. The purpose of this study was to investigate the capability of the genetically engineered Salmonella typhimurium (S. typhimurium) to access the glioma xenograft, which was monitored in mouse brain tumor models using optical bioluminescence imaging technique. METHODS: U87 malignant glioma cells (U87-MG) stably transfected with firefly luciferase (Fluc) were implanted into BALB/cAnN nude mice by stereotactic injection into the striatum. After tumor formation, attenuated S. typhimurium expressing bacterial luciferase (Lux) was injected into the tail vein. Bioluminescence signals from transfected cells or bacteria were monitored using a cooled charge-coupled device camera to identify the tumor location or to trace the bacterial migration. Immunofluorescence staining was also performed in frozen sections of mouse glioma xenograft. RESULTS: The injected S. typhimurium exclusively localized in the glioma xenograft region of U87-MG-bearing mouse. Immunofluorescence staining also demonstrated the accumulation of S. typhimurium in the brain tumors. CONCLUSION: The present study demonstrated that S. typhimurium can target glioma xenograft, and may provide a potentially therapeutic probe for glioma.
Animals ; Bacteria ; Brain Neoplasms ; Fireflies ; Fluorescent Antibody Technique ; Frozen Sections ; Glioma* ; Heterografts ; Injections, Intravenous ; Luciferases ; Mice* ; Mice, Nude ; Salmonella ; Salmonella typhimurium* ; Veins

A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.
Animals ; Base Sequence ; Cricetinae ; Crustacea ; enzymology ; Fireflies ; enzymology ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. The application of recombinant reporter plasmid such as the firefly luciferase gene has proven to be a very effective method to detect these chemicals. The bioassay system, CALUX, is sensitive in directly detecting AhR-agonists from a variety of environmental and biologic materials. However, responses of the AhR-dependent bioassays are dependent on the cell types used. Thus, we developed a sensitive bioassay using the recombinant mouse hepatoma cell (Hepa1c1c7) for the determination of dioxins. The recombinant cell line was stably transfected with firefly luciferase reporter gene (pGudLuc1.1). The transfected cells showed the highest induction of luciferase activity at 4.5 hr and a decrease beyond this time point. The system showed the highest sensitivity of detection ever reported. Upon TCDD exposure cells showed 2 fold increase at 10 pM and 7 fold increase at 100 pM, respectively. The passage number after the transfection played an important role in the sensitivity. The increase of passage number tended to increase the sensitivity of the cells up to 15. The media without phenol red showed a higher induction rate than with phenol red, suggesting the preferable use of phenol red-free media for the bioassay. Since each of the assays has unique characteristics that make them suitable for some screening applications and not others, development of sensitive bioanalytical methods based on a variety of cellular systems in a key to the successful determination of dioxins. The bioassay system developed in this study will contribute to further development of successful screening the AhR agonists among the environmental mixture. In addition, the rapid and sensitive nature of this cellular system can be applied as a valuable tool to screen the dioxin-like moieties among the prodrugs at the initial stage, thereby expediting the new drug discovery.
Animals ; Biological Assay* ; Carcinoma, Hepatocellular ; Cell Line ; Dioxins ; Drug Discovery ; Fireflies ; Genes, Reporter ; Luciferases* ; Mass Screening ; Mice ; Phenol ; Phenolsulfonphthalein ; Plasmids ; Prodrugs ; Tetrachlorodibenzodioxin ; Transcription Factors ; Transfection