Pushdown automata in statistical machine translation

This article describes the use of pushdown automata (PDA) in the context of statistical machine translation and alignment under a synchronous context-free grammar. We use PDAs to compactly represent the space of candidate translations generated by the grammar when applied to an input sentence. General-purpose PDA algorithms for replacement, composition, shortest path, and expansion are presented. We describe HiPDT, a hierarchical phrase-based decoder using the PDA representation and these algorithms. We contrast the complexity of this decoder with a decoder based on a finite state automata representation, showing that PDAs provide a more suitable framework to achieve exact decoding for larger synchronous context-free grammars and smaller language models. We assess this experimentally on a large-scale Chinese-to-English alignment and translation task. In translation, we propose a two-pass decoding strategy involving a weaker language model in the first-pass to address the results of PDA complexity analysis. We study in depth the experimental conditions and tradeoffs in which HiPDT can achieve state-of-the-art performance for large-scale SMT. </jats:p

TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 μM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination

Structures of the two most promising compounds.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 μM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.</div

Translation of C-ELISA-HRP to the HTS platform.

(A) Europium conjugated secondary antibodies can be used for the cell-based assay. C-ELISA was performed with Europium conjugated rabbit IgG at different dilutions. The error bars represent the ± SEM of three wells in one experiment (*Z’ factor > 0.5). (B, C) The washing steps were done either manually (B) or with an automated wash system (C). The error bars represent the ± SEM of three wells in one experiment (* Z’ factor > 0.5). (D) C-ELISA-Eu detects the increase in α-tubulin nitrotyrosination with time. The error bars represent the ± SEM of three wells in one experiment (*Z’ factor > 0.5). (E, F) Washing steps with PBS can be replaced with the DelphiaTM buffers used for the C_ELISA-Eu. (E) Washing steps before the addition of rabbit IgG were done with PBS, and DELPHIATM buffer was used thereafter. (F) All the washing steps were performed with DELPHIATM buffer. The error bars represent the ± SEM of three wells in one experiment. The y-axis represents the time-resolved fluorescence values at emission/excitation wave lengths of 340/615 nm. The symbol * indicates Z’ factors > 0.5. (TIF)</p

Effect of paclitaxel and nocodazole on cell viability and α-tubulin nitrotyrosination.

(A) Growth curves with paclitaxel. HEp-2 cells were treated with nitrotyrosine (100 μM), paclitaxel (100 nM), both or neither (untreated). Viable cells were counted using the tryphan blue exclusion assay and plotted as cells per well against time. The error bars represent the ± SEM of two independent experiments with duplicates in each experiment. (B) Nitrotyrosinated α-tubulin levels with paclitaxel. HEp-2 cells were treated as in A. Total cell lysates were made at the stated times and analyzed by immunoblotting with anti-nitrotyrosine, anti-α-tubulin and anti-TBP antibodies. A representative immunoblot is shown. (C) Growth curves with nocodazole. HEp-2 cells were treated with nitrotyrosine (100 μM), nocodazole (1 or 10 nM), both or neither (untreated, with the equivalent amount of solvents). Viable cells were counted using the tryphan blue exclusion assay and cells per well plotted against time. The error bars represent the ± SEM of two independent experiments with duplicates in each experiment. (D) Nitrotyrosinated α-tubulin levels with nocodazole. HEp-2 cells were treated as in A. Total cell lysates were made at stated time intervals and analyzed by immunoblotting with anti-nitrotyrosine, anti-TBP or anti α-tubulin antibodies. A typical immunoblot is shown.</p

Schematic summary.

The diagram illustrates the detyrosination-tyrosination cycle of α-tubulin, in which TCP and TTL catalyse the detyrosination and religation of tyrosine (Y) to α-tubulin. Under oxidative (Oxy.) stress nitrotyrosine (NO2-Y) is ligated to detyrosinated α-tubulin (see box) to give nitrotyrosinated α-tubulin, which has been hypothesised to lead to cell death and suppression of this pathway could lead to oncogenesis. TTTLL12 inhibits ligation of nitrotyrosine to detyrosinated α-tubulin, which is a potential oncogenic activity. Inhibition of this process by small molecule inhibitors could lead to novel therapeutic drugs.</p