Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion

EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) is involved in multiple aspects of cancer cell biology. EGFR has already been identified as an important target for cancer therapy, with various kinds of EGFR inhibitors currently used in treatment of several human cancers. Recently, EGFR and its downstream signaling pathways were identified as being associated with cisplatin sensitivity. In addition, EGFR inhibitors have shown significant promise for patients who failed cisplatin-based therapy. In this study, we investigated whether treatment with an EGFR inhibitor improves cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell lines. The effects of a combination of AG1478, a specific EGFR tyrosine kinase inhibitor, with cisplatin were evaluated in cultured OSCC cell lines and cisplatin-resistant sublines. Higher expression of EGFR and p-EGFR was found in the two cisplatin-resistant cell lines compared with the corresponding parental cell lines. In addition, augmented inhibition of OSCC cell growth by the combination of AG1478 with cisplatin was found in both cell lines. These results suggest that the combination of an EGFR inhibitor and cisplatin may be useful as a rational strategy for the treatment of patients with oral cancer with acquired cisplatin resistance

Expression of ADAMTS-5 in deformed human temporomandibular joint discs

Objective: To study the expression of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in tissue samples of deformed human temporomandibular joint (TMJ) discs and cells obtained from the discs. Materials and methods: Eleven adult human TMJ discs (nine diseased discs and two normal discs) were used in this study. The nine diseased discs were obtained from nine patients with internal derangement (ID) and osteoarthritis (OA) in the TMJ. These patients all had anteriorly displaced discs and deformed mandibular condyles, making conservative therapy impossible. The tissues were immunohistochemically stained using ADAMTS-5 antibodies. In addition, an articular disc cell line from one case was established by collagenase treatment. The subcultured cells under both normal and hypoxic conditions (O2: 2%) were incubated for 3, 6, 12 and 24 h after addition of interleukin-1ß (IL-1ß) (1 ng/mL). Subsequently, the expression of ADAMTS-5 was examined using reverse transcription-polymerase chain reaction (RT-PCR). Results: The control group showed negative reactions on immunohistochemical staining. The discs extracted from cases with ID and OA presented positive reactions for ADAMTS-5. The expression of ADAMTS-5 mRNA increased under both normoxia and hypoxia with the addition of IL-1ß, and the peak was observed after 3 h. Conclusion: These results suggest that ADAMTS-5 is related to deformation and destruction of human TMJ discs affected by ID and OA

Characterization of ash deposition and heat transfer behavior of coals during combustion in a pilot-scale facility and full-scale utility

Experimental measurements as well as theoretical models were used to investigate the impact of mineral matter of three coals on ash deposition and heat transfer for pulverized coal fired boilers. The ash deposition experiments were conducted in a pulverized fuel combustion pilot-scale facility and a full-scale unit. A mathematical model with input from computer-controlled scanning electron microscopy analysis of coal minerals was used to predict the effect of ash deposition on heat transfer. The predicted deposit thickness and heat flux from the model are shown to be consistent with the measurements in the test facility. The model differentiates the coals according to the deposits they form and their effect on heat transfer. The heat transfer predictions in the full-scale unit were found to be most suitable for the water wall under the furnace nose. The study demonstrates that the measurements in a full-scale unit can differ significantly from those in pilot-scale furnaces due to soot-blowing operations

General Applicability of Synthetic Gene-Overexpression for Cell-Type Ratio Control via Reprogramming

Control of the cell-type ratio in multistable systems requires wide-range control of the initial states of cells. Here, using a synthetic circuit in <i>E. coli</i>, we describe the use of a simple gene-overexpression system combined with a bistable toggle switch, for the purposes of enabling the wide-range control of cellular states and thus generating arbitrary cell-type ratios. Theoretically, overexpression induction temporarily alters the bistable system to a monostable system, in which the location of the single steady state of cells can be manipulated over a wide range by regulating the overexpression levels. This induced cellular state becomes the initial state of the basal bistable system upon overexpression cessation, which restores the original bistable system. We experimentally demonstrated that the overexpression induced a monomodal cell distribution, and subsequent overexpression withdrawal generated a bimodal distribution. Furthermore, as designed theoretically, regulating the overexpression levels by adjusting the concentrations of small molecules generated arbitrary cell-type ratios