Development of Terminator-Promoter Bifunctional Elements for Application in Saccharomyces cerevisiae Pathway Engineering.

The construction of a genetic circuit requires the substitution and redesign of different promoters and terminators. The assembly efficiency of exogenous pathways will also decrease significantly when the number of regulatory elements and genes is increased. We speculated that a novel bifunctional element with promoter and terminator functions could be created via the fusion of a termination signal with a promoter sequence. In this study, the elements from a Saccharomyces cerevisiae promoter and terminator were employed to design a synthetic bifunctional element. The promoter strength of the synthetic element is apparently regulated through a spacer sequence and an upstream activating sequence (UAS) with a ~5-fold increase, and the terminator strength could be finely regulated by the efficiency element, with a ~5-fold increase. Furthermore, the use of a TATA box-like sequence resulted in the adequate execution of both functions of the TATA box and the efficiency element. By regulating the TATA box-like sequence, UAS, and spacer sequence, the strengths of the promoter-like and terminator-like bifunctional elements were optimally fine-tuned with ~8-fold and ~7-fold increases, respectively. The application of bifunctional elements in the lycopene biosynthetic pathway showed an improved pathway assembly efficiency and higher lycopene yield. The designed bifunctional elements effectively simplified pathway construction and can serve as a useful toolbox for yeast synthetic biology.

Carotenoid Biosynthesis: Genome-Wide Profiling, Pathway Identification in Rhodotorula glutinis X-20, and High-Level Production.

Rhodotorula glutinis, as a member of the family Sporidiobolaceae, is of great value in the field of biotechnology. However, the evolutionary relationship of R. glutinis X-20 with Rhodosporidiobolus, Sporobolomyces, and Rhodotorula are not well understood, and its metabolic pathways such as carotenoid biosynthesis are not well resolved. Here, genome sequencing and comparative genome techniques were employed to improve the understanding of R. glutinis X-20. Phytoene desaturase (crtI) and 15-cis-phytoene synthase/lycopene beta-cyclase (crtYB), key enzymes in carotenoid pathway from R. glutinis X-20 were more efficiently expressed in S. cerevisiae INVSc1 than in S. cerevisiae CEN.PK2-1C. High yielding engineered strains were obtained by using synthetic biology technology constructing carotenoid pathway in S. cerevisiae and optimizing the precursor supply after fed-batch fermentation with palmitic acid supplementation. Genome sequencing analysis and metabolite identification has enhanced the understanding of evolutionary relationships and metabolic pathways in R. glutinis X-20, while heterologous construction of carotenoid pathway has facilitated its industrial application.

Insights from the proteome profile of Phytophthora capsici in response to the novel fungicide SYP-14288.

Phytophthora capsica is a destructive oomycete plant pathogen that causes huge losses to crop production worldwide. However, the novel fungicide SYP-14288 has shown excellent activity against various stages of the oomycete life cycle as well against fungal plant pathogens. The current study utilized isobaric tags for relative and absolute quantitation technology to generate proteome profiles of P. capsici in the presence or absence of SYP-14288 in order to gain a greater understanding of the SYP-14288 mode of action. A total of 1,443 individual proteins were identified during the investigation, of which 599 were considered to have significantly altered expression. Further investigation using Cluster of Orthologous Groups of proteins analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated most of the proteins with altered expression were associated with carbohydrate metabolism, energy metabolism and their downstream biological functions, especially with regard to oxidoreductase activity and subsequent adenosine triphosphate (ATP) production associated pathways. Quantitative expression analysis using qRT-PCR validated the proteomic data. These results seem to indicate that SYP-14288 treatment caused a shift in energy metabolism that resulted in the activation of compensatory mechanisms affecting carbohydrate and lipid metabolism. The study also found evidence that the up-regulation of transmembrane transporters and proteins associated with stress response might also be coopted to compensate for the disrupted proton gradient and other downstream effects. Taken together these results provide strong evidence that SYP-14288 has a similar mode of action to the oxidative phosphorylation uncoupler Fluazinam but further investigation, including molecular studies, is required to completely characterize the SYP-14288 mode of action in P. capsici. However, the proteomic data collected in the current study does provide important insight into the overall effect of SYP-14288 in P. capsici, which could be useful for the registration and application of this novel fungicide.

The novel fungicide SYP-14288 acts as an uncoupler against Phytophthora capsici.

SYP-14288 is a novel fungicide developed by the Shenyang Research Institute of Chemical Industry in China. Although preliminary studies indicate that SYP-14288 is highly effective against 32 important plant pathogens belonging to a range of taxonomic groups, its mode of action remains unknown. In this study, we documented that SYP-14288 has excellent activity against all of the asexual life stages of the plant-pathogenic oomycete Phytophthora capsici, and is especially effective in blocking cyst germination and other life stages that require high energy consumption. In assays designed to determine the fungicide's mode of action, addition of ATP reduced SYP-14288 inhibition of P. capsici, which suggested that SYP-14288 inhibits ATP synthesis of the pathogen. This inference was confirmed in that treatment with SYP-14288 sharply reduced the ATP content in P. capsici. The respiration rate of P. capsici was positively correlated with the concentration of SYP-14288 or of the fungicide fluazinam (an uncoupler of oxidative phosphorylation), but increases in respiration were greater with SYP-14288 than with fluazinam. These results indicate that SYP-14288 is a promising fungicide that functions as an uncoupler of oxidative phosphorylation.

Comparison of ablation zone between 915- and 2,450-MHz cooled-shaft microwave antenna: results in in vivo porcine livers.

OBJECTIVE: The objective of our study was to compare the ablation zones of two cooled-shaft microwave antennae-one operating at 915 MHz and one operating at 2,450 MHz-in in vivo porcine livers. CONCLUSION: The 915-MHz cooled-shaft microwave antenna can lead to deeper penetration of liver tissue and can yield a significantly larger ablation zone than the 2,450-MHz cooled-shaft antenna in in vivo porcine livers. In light of the result, 915-MHz cooled-shaft microwaves may be more effective for tumor ablation, especially for large tumors.

Ultrasound guided percutaneous microwave ablation for small renal cancer: initial experience.

PURPOSE: We evaluated the feasibility, safety and efficacy of ultrasound guided percutaneous microwave ablation for small renal cell cancers. MATERIALS AND METHODS: A total of 12 patients with a pathologically proven renal cell cancer 1.3 to 3.8 cm in diameter were treated with microwave ablation. A cooled shaft needle antenna was percutaneously inserted into the tumor under ultrasound guidance. One antenna was used for tumors 2 cm or smaller and antennae were used for tumors larger than 2 cm. One thermocouple was placed about 0.5 cm away from the tumor to monitor temperature in real time during ablation. Microwaves were emitted at 50 W for 500 seconds and prolonged as necessary to attain temperatures sufficient to ensure tumor kill. Immediate treatment efficacy was assessed by contrast enhanced ultrasound 1 day after ablation. Short-term efficacy was assessed by contrast enhanced computerized tomography and/or contrast enhanced ultrasound at 1, 3 and 6 months, and every 6 months thereafter. RESULTS: All tumors were completely ablated at a single session and no complications occurred. No residual tumor or recurrence was observed at a median followup of 11 months (range 4 to 20). The ablation zone was well defined on contrast enhanced imaging and it gradually shrank with time. CONCLUSIONS: Ultrasound guided percutaneous microwave ablation appears to be a safe and effective technique for small renal cell cancer in select patients.

Internally cooled antenna for microwave ablation: results in ex vivo and in vivo porcine livers.

PURPOSE: To evaluate the performance of microwave ablation with an internally cooled antenna in ex vivo and in vivo porcine livers. MATERIALS AND METHODS: Microwave ablations were performed in ex vivo and in vivo porcine livers with a 1.9 mm-diameter 2450 MHz microwave needle antenna. Four power settings (50, 60, 70 and 80 W) were used in this study, while application time was fixed at 10 min. Diameters of the coagulation zone were observed on gross specimens. RESULTS: Excessive elongation of coagulation along the needle shaft was not encountered in all ablations. An arrow-shaped charring was observed. As the power increases, the long-axis coagulation diameter for in vivo liver increased significantly (P<.05). The short-axis coagulation diameter for in vivo liver was significantly smaller than that for ex vivo liver (P<.05) but not statistically different among the four power settings. After 10 min of ablation at 80 W, the short-axis and long-axis coagulation diameter for in vivo liver was 4.92+/-0.15 cm and 2.37+/-0.10 cm, respectively. CONCLUSION: The internally cooled microwave antenna may be advantageous to minimize collateral damages.

[Intra-nasal scanning of tumors in nasal cavity and paranasal sinus with endoscopic ultrasonography].

OBJECTIVE: To evaluate endoscopic ultrasonography for masses in nasal cavity and paranasal sinus. METHODS: Under the guidance of nasal endoscope, sonographic scan of 18 masses within nasal cavity and paranasal sinus was performed by using 10 MHz catheter transducer with diameter of 3.3 mm under local anesthesia. Twelve of them were benign tumors and 6 of them were malignant ones, which were confirmed by pathological examination of resected specimens. RESULTS: Under the guidance of nasal endoscope, masses could be observed accurately with catheter transducer. On gray scale ultrasound, most masses were heterogeneous hypoechoic, tumors with rich blood vessels were lower hypoechoic, and some showed irregular anechoic area due to dilated vascular net. Neurofibroma was with well-defined and regular border and entire capsule; chordoma was without distinct edge and capsule. A giant pituitary tumor eroding bone of sphenoid sinus and intruding into nasal cavity. The relationship between mass and internal carotid artery could be demonstrated using color Doppler flow imaging (CDFI). Blood flowing signals in masses could be detected by CDFI, and spectral Doppler could discriminate arterial or venous blood flowing signals and measure its velocity. The rich blood supply was observed in fibroangioma, the rich flow signals and high velocity could be detected in malignant tumors. CONCLUSIONS: Nasal endoscope-guided sonography for soft tissue masses in nasal cavity is of exact location, clear image and high resolution, which can reveal blood flow signals sensitively, differentiate arterial and venous blood signals and measure the velocity of them. It provides a new imaging modality for masses within nasal cavity, sinuses and skull base.