Early Experience and the Feasibility of the Firefly Technology for Central Lymph Node Dissection (CLND) in Robotic Thyroidectomy Using the BABA

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.

Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

Targeting Orthotopic Glioma in Mice with Genetically Engineered Salmonella typhimurium

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.

Expression of thermostable recombiant Luciola lateralis luciferase and development of heat-stable pyrosequencing system / 生物工程学报

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.

Construction of a dual-luciferase co-expression vector and its characteristics in vitro and in vivo / 病毒学报

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.

Description of Coctilelater minimus sp. nov. (Coleoptera, Elateridae, Agrypninae) / Descrição de Coctilelater minimus sp. nov. (Coleoptera, Elateridae, Agrypninae)

Coctilelater minimus from Brazil (Pará) is described and illustrated. This new species is mainly characterized by small size and coloration pattern.

Coctilelater minimus sp. nov., coletado no Brasil (Pará) é descrita e ilustrada. Essa espécie é caracterizada principalmente pelo pequeno tamanho e pela coloração geral do corpo.

Metapyrophorus pharolim a new genus and species of Pyrophorini (Coleoptera, Elateridae, Agrypninae) / Metapyrophorus pharolim, gênero e espécie novos de Pyrophorini (Coleoptera, Elateridae, Agrypninae)

Metapyrophorus, a new monotypic genus, is erected based on M. pharolim, new species from Trinidad and Tobago and Venezuela. The genus is characterized mainly by its pair of convex pronotal bioluminescent organs, equidistant between the median line and the lateral margin.

O gênero monotípico Metapyrophorus é erigido com base na nova espécie M. pharolim, de Trinidad e Tobago e Venezuela. O gênero é caracterizado principalmente por um par de órgãos bioluminescentes convexos, localizados próximos da margem posterior do pronoto, eqüidistantes à linha mediana e à margem lateral.

Description of Photuris fulvipes (Blanchard) immatures (Coleoptera, Lampyridae, Photurinae) and bionomic aspects under laboratory conditions

Larvae and pupae of the cryptic firefly Photuris fulvipes (Blanchard,1837) are described. They were reared to adult stage from eggs laid by a female collected in São Luiz do Paraitinga, São Paulo, Brazil. Additionally, adult diagnosis and some bionomic data observed in the laboratory are presented. The bionomical and morphological characteristics of P. fulvipes were compared to other unidentified species of the genus and to Bycellonycha sp. from literature. At moment, the data are insufficient to propose a diagnosis for the species, but some characteristics appear to define species group or, at least, they may have generic value.

Larva e pupa de Photuris fulvipes (Blanchard, 1837), criadas a partir de ovos de uma fêmea coletada em São Luiz do Paraitinga, São Paulo, são descritas. São apresentados uma diagnose do adulto e alguns dados bionômicos observados durante a criação em laboratório. Esses dados e a morfologia dos imaturos foram comparados aos de outras espécies não identificadas do gênero e a Bycellonycha sp., descritos na literatura. Nesse momento, os dados são insuficientes para propor uma diagnose para a espécie, mas algumas características parecem ser importantes para definir grupos de espécies ou, pelo menos, para diagnosticar o gênero.

Effects of Intracavernosally Injected Embryonic Stem Cells in Aged Rat / 대한남성과학회지

PURPOSE: Stem cell-based cell therapy has recently been tried as a way to restore cavernosal function in an animal model. The aims of this study were to elucidate the effect of intracavernosally injected embryonic stem cells(ESCs) in aged rat. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into 3 groups: young control(12 weeks old; n=10), old with vehicle injection(24 months old; n=7), and old with ESC injection(24 months old; n=8). ESCs were transfected with firefly luciferase attached to adenovirus and then injected intracavernously 2 times with a 1-week interval. Cell survival was assessed by optical molecular imaging 2 days after the last ESC injection. At 4 weeks after the last injection, intracavernosal pressure and systemic arterial pressure were recorded after pelvic nerve stimulation. Serum testosterone levels were measured by radioimmunoassay. RESULTS: We observed fluorescent signals around the external genitalia of animals injected with ESCs. The serum testosterone level of the old group(1.39+/-0.07 ng/ml) was significantly lower compared to the young control group(2.98+/-0.31 ng/ml)(p=0.03). The percentage of intracavernosal pressure/systolic blood pressure was significantly lower in the old group(58.5+/-8.6%) compared to the young control group(69.5+/-6.6%)(p=0.034). However, the old group with ESC injection(61.6+/-9.9%) did not show any significant differences from the old group(p>0.05). The old group with ESC injection showed histomorphometry similar to the old group. CONCLUSIONS: The presence of intracavernosal ESCs can be noninvasively monitored with optical molecular imaging. However, the intracavernosal injection of ESCs did not improve erectile function in the aging rat. Further studies are needed to elucidate the regulatory factors of stem cell differentiation in the corpus cavernosum.

Bioluminescence Imaging of Chondrocytes in Rabbits by Intraarticular Injection of D-Luciferin / 핵의학분자영상

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.

Development of Luciferase Reporter Gene-based Cell Bioassay for the Aromatic Hydrocarbon Receptor Agonists

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.

Effects of Testosterone on the Intracavernosally Injected Embryonic Stem Cells in Rat / 대한남성과학회지

PURPOSE: Stem cell-based cell therapy has recently been tried as a way to restore cavernosal function in an animal model. The aim of this study was to elucidate the role of testosterone on intracavernosally injected embryonic stem cells (ESCs) in rat. MATERIALS AND METHODS: Male Sprague Dawley rats(12 weeks old; n=25) were divided into five groups: control, castration, ESC injection after castration, testosterone supplementation after castration, and ESC injection with testosterone supplementation after castration(n=5 in each group). ESCs were transfected with firefly luciferase attached to adenovirus and then injected intracavernously. Testosterone propionate(0.1 mg) was subcutaneously injected daily in the testosterone supplementation group. Cell survival was assessed by optical molecular imaging the day after the ESC injection. After 4 weeks of treatment, intracavernosal pressure and systemic arterial pressure were recorded after pelvic nerve stimulation. Serum testosterone levels were measured by radioimmunoassay. RESULTS: With optical molecular imaging, we observed fluorescent signals around the external genitalia of all ESC-injected animals. The percentage of intracavernosal pressure/systolic blood pressure was significantly lower in the castration group(15.9+/-5.3%) compared to the control group(53.7+/-9.7%)(p0.05). The ESC injection with testosterone supplementation group(54.2+/-4.3%) showed erectile function similar to the control group. CONCLUSIONS: Intracavernosal injection of embryonic stem cells did not improve erectile function in the castrated rat. This result implies that testosterone may play an essential role in the proliferation of stem cells in the corpus cavernosum.

Effects of Testosterone on the Intracavernosally Injected Embryonic Stem Cells in Rat / 대한남성과학회지

PURPOSE: Stem cell-based cell therapy has recently been tried as a way to restore cavernosal function in an animal model. The aim of this study was to elucidate the role of testosterone on intracavernosally injected embryonic stem cells (ESCs) in rat. MATERIALS AND METHODS: Male Sprague Dawley rats(12 weeks old; n=25) were divided into five groups: control, castration, ESC injection after castration, testosterone supplementation after castration, and ESC injection with testosterone supplementation after castration(n=5 in each group). ESCs were transfected with firefly luciferase attached to adenovirus and then injected intracavernously. Testosterone propionate(0.1 mg) was subcutaneously injected daily in the testosterone supplementation group. Cell survival was assessed by optical molecular imaging the day after the ESC injection. After 4 weeks of treatment, intracavernosal pressure and systemic arterial pressure were recorded after pelvic nerve stimulation. Serum testosterone levels were measured by radioimmunoassay. RESULTS: With optical molecular imaging, we observed fluorescent signals around the external genitalia of all ESC-injected animals. The percentage of intracavernosal pressure/systolic blood pressure was significantly lower in the castration group(15.9+/-5.3%) compared to the control group(53.7+/-9.7%)(p0.05). The ESC injection with testosterone supplementation group(54.2+/-4.3%) showed erectile function similar to the control group. CONCLUSIONS: Intracavernosal injection of embryonic stem cells did not improve erectile function in the castrated rat. This result implies that testosterone may play an essential role in the proliferation of stem cells in the corpus cavernosum.

Functional Modification of a Specific RNA with Targeted Trans-Splicing

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.

Marker Gene Transfer into Cells of Rat Achilles Tendon by In Vivo Electroporation / 대한정형외과학회잡지

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.

Genetic Variants of Thromobomodulin Gene as Risk Factors for Myocardial Infarction

Thrombomodulin (TM) is thrombin receptor present on the luminal surface of endothelial cells. Because the thrombin-TM complex acts as an anticoagulant, the functional variants or deficiency of TM may lead to increment of thrombotic tendency. In this study, we screened the genetic variants of the TM gene in patients with myocardial infarction (MI) and analyzed the genotype to elucidate the effects of genetic variations of TM gene on the development of the MI. We screened a promoter region and coding sequence of the TM gene using single strand conformation polymorphism-heteroduplex analysis and identified three common genetic variants: those were TM G-33A, TM Ala455Val, and TM C1922T. The genotype frequencies were investigated in the patients with MI (n=234) and control subjects (n=291) by the method of allele-specific oligomer hybridization. The frequencies of mutant genotypes (TM -33A, TM 455Val, and TM 1922T) were higher in patient group compared to the control subjects in males while there were no significant differences in females. In the multiple logistic regression analysis, TM 455Val and TM 1922T alleles were independent risk factors for MI (OR[95% CI: 1.799[1.125-2.878] p=0.014 and 5.624[1.019-31.025], p=0.048, respectively) in males. However, the genetic variations were not independent risk factors for MI in females. There were significant linkage disequilibriums among three genetic variants. These linkage disequilibriums explain the similar effects of three genetic variants on the development of MI. To investigate the effect of the TM G-33A mutation on TM promoter activity, the two TM promoter constructs (pTM-355 and pTM-125, bearing TM -33G or TM -33A) containing of firefly luciferase gene were transfected into HepG2, BAE, and CHO cells. The promoter activities were higher in the promoter constructs with TM -33G compared to the constructs with TM -33A in pTM-355. These results suggest the possibility of the positive predisposing effect of TM -33A allele on MI in males. The functional study for TM Ala455Val and TM C1922T should be followed to elucidate the genotype effects of these mutations on the development of MI. In this study, we identified three genetic variants of TM gene and showed the significant associations between genetic variants and MI in males. These results proposed that TM gene is an attractive candidate for genetic risk factor for MI in Koreans.

The Change of c-jun Promoter Activity in TPA-Induced U937 Cells Infected with Human Cytomegalovirus (HCMV)

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.

The Change of c-jun Promoter Activity in TPA-Induced U937 Cells Infected with Human Cytomegalovirus (HCMV)

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.