Sun-Ha Park

Ablative microstructuring with plasma-based XUV lasers and efficient processing of materials by dual action of XUV/NIR–VIS ultrashort pulses

We report on a single-shot micropatterning of an organic polymer achieved by ablation with demagnifying projection using a plasma-based extreme ultraviolet (XUV) laser at 21 nm. A nickel mesh with a period of 100 μ m was 10×demagnified and imprinted on poly(methyl methacrylate) (PMMA) via direct ablation. This first demonstration of single-shot projection, single-step lithography illustrates the great potential of XUV lasers for the direct patterning of materials with a resolution scalable down to the submicrometer domain. In the second part, we present a novel experimental method for improving the efficiency of surface processing of various solids achieved by simultaneous action of XUV, obtained from high-order harmonic generation, and near-infrared (NIR)–VIS laser pulses. The NIR–VIS pulse interacts with free charge carriers produced by the energetic XUV photons, so that its absorption dramatically increases. Laser-induced periodic surface structures were effectively produced using this technique.

Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum.

A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.

Crystal structure and functional characterization of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea strain 34H.

Isoaspartyl dipeptidase (IadA) is an enzyme that catalyzes the hydrolysis of an isoaspartyl dipeptide-like moiety, which can be inappropriately formed in proteins, between the β-carboxyl group side chain of Asp and the amino group of the following amino acid. Here, we have determined the structures of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea, both ligand-free and that complexed with β-isoaspartyl lysine, at 1.85-Å and 2.33-Å resolution, respectively. In both structures, CpsIadA formed an octamer with two Zn ions in the active site. A structural comparison with Escherichia coli isoaspartyl dipeptidase (EcoIadA) revealed a major difference in the structure of the active site. For metal ion coordination, CpsIadA has a Glu166 residue in the active site, whereas EcoIadA has a post-translationally carbamylated-lysine 162 residue. Site-directed mutagenesis studies confirmed that the Glu166 residue is critical for CpsIadA enzymatic activity. This residue substitution from lysine to glutamate induces the protrusion of the β12-α8 loop into the active site to compensate for the loss of length of the side chain. In addition, the α3-β9 loop of CpsIadA adopts a different conformation compared to EcoIadA, which induces a change in the structure of the substrate-binding pocket. Despite CpsIadA having a different active-site residue composition and substrate-binding pocket, there is only a slight difference in CpsIadA substrate specificity compared with EcoIadA. Comparative sequence analysis classified IadA-containing bacteria and archaea into two groups based on the active-site residue composition, with Type I IadAs having a glutamate residue and Type II IadAs having a carbamylated-lysine residue. CpsIadA has maximal activity at pH 8–8.5 and 45°C, and was completely inactivated at 60°C. Despite being isolated from a psychrophilic bacteria, CpsIadA is thermostable probably owing to its octameric structure. This is the first conclusive description of the structure and properties of a Type I IadA.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier

The two-component phosphorelay system is the most prevalent mechanism for sensing and transducing environmental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long-term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key response regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regulatory N-terminal phospho-receiver domain and a DNA-binding C-terminal activator domain. We solved the three-dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearothermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the mechanism of phosphorylation-dependent activation unique to Spo0A.

Efficient surface processing by ultrafast XUV/NIR dual action

We have developed a novel method for efficient structuring of the surface of materials by applying femtosecond near infrared laser pulses simultaneously with a weak extreme ultraviolet beam, which leads to a very strong radiation-matter interaction and brings a dramatic increase of the surface processing speed. We present our recent experimental results on surface nanostructuring of thin films of amorphous carbon and polymethyl methacrylate deposited on bulk substrates and discuss the underlying physical mechanisms. In the case of amorphous carbon, large areas of laser-induced periodic surface structures with a spatial period of 550 nm were created, having their origin in laser-induced convective currents. Our method provides a powerful tool for fast modification of tribological properties of the irradiated sample.

Highly Efficient Surface Modification of Solids by Dual Action of XUV/Vis-NIR Laser Pulses

We demonstrate a novel experimental method for unusual structural surface modification of various solids (PMMA, amorphous carbon) achieved by simultaneous action of XUV (21.6 nm), obtained from high-order harmonic generation, and Vis-NIR (410/820 nm) laser pulses. Although the fluence of each individual pulse was far below the surface ablation threshold, very efficient and specific material expansion was observed after irradiation by a few shots of mixed XUV/Vis-NIR radiation.

Crystal structure of dihydrodipicolinate reductase (PaDHDPR) from Paenisporosarcina sp. TG-14: structural basis for NADPH preference as a cofactor

Dihydrodipicolinate reductase (DHDPR) is a key enzyme in the diaminopimelate- and lysine-synthesis pathways that reduces DHDP to tetrahydrodipicolinate. Although DHDPR uses both NADPH and NADH as a cofactor, the structural basis for cofactor specificity and preference remains unclear. Here, we report that Paenisporosarcina sp. TG-14 PaDHDPR has a strong preference for NADPH over NADH, as determined by isothermal titration calorimetry and enzymatic activity assays. We determined the crystal structures of PaDHDPR alone, with its competitive inhibitor (dipicolinate), and the ternary complex of the enzyme with dipicolinate and NADPH, with results showing that only the ternary complex had a fully closed conformation and suggesting that binding of both substrate and nucleotide cofactor is required for enzymatic activity. Moreover, NADPH binding induced local conformational changes in the N-terminal long loop (residues 34–59) of PaDHDPR, as the His35 and Lys36 residues in this loop interacted with the 2′-phosphate group of NADPH, possibly accounting for the strong preference of PaDHDPR for NADPH. Mutation of these residues revealed reduced NADPH binding and enzymatic activity, confirming their importance in NADPH binding. These findings provide insight into the mechanism of action and cofactor selectivity of this important bacterial enzyme.

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10–C12 n-alkanes and C10–C14 fatty acids with Kd values varied from 0.42 to 0.59 μM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the β1–β2, α3–α4, and α6–α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.

Abstract P4-12-28: HER2-related biomarkers in HER2+ breast cancer patients in Asia Pacific

Recent findings suggest that HER2-related molecular markers such as PTEN deletion or downregulation, PIK3CA mutation, truncated HER2 receptor (p95HER2), and tumor HER2 mRNA levels, have the potential to predict anti-HER2 treatment response. We evaluated the distribution of these biomarkers at the time of primary diagnosis and their relationship to responsiveness to lapatinib treatment in the metastatic setting in HER2+ breast cancer patients.nnWe conducted an observational study of female HER2+ breast cancer patients who were initiated on lapatinib treatment following recurrence or metastases in five Asia Pacific countries. Patients were enrolled between August 2010 and December 2012. Eligible patients had a tumor biopsy specimen available from their primary breast cancer diagnosis or before they started on any anti-HER2 treatment, had not been exposed to more than two lines of anti-HER2 treatment in the metastatic setting or other experimental anti-HER2 treatment, and had no other primary tumor. Biomarkers levels at primary diagnosis were measured; PTEN levels were assessed by immunohistochemistry and PIK3CA mutations were detected by a mass spectroscopy-based approach. The primary endpoint was progression-free survival (PFS) from the initiation of first lapatinib-based regimen given in metastatic setting to disease progression from that regimen or death from any cause. PFS analysis was conducted with a data cut-off date of 31 December 2012.nnA total of 162 patients were included in this study and 96% have confirmed HER2+ breast cancer primary tumor. The mean age was 52±10 years and 97% had metastases at study entry, with bone being the most common site of metastasis (48%). About a quarter had PTEN protein loss (24%), 30% had PIK3CA mutation, and 7% had both at primary diagnosis. No significant association was observed between both biomarkers or between each biomarker and estrogen receptor status or HER2 status.nnView this table:nnTable 1. Relationship between PTEN and PIK3CAnnnnnnPatients with altered PTEN expression, or PIK3CA mutation showed comparable PFS with lapatinib-based treatment as those with normal PTEN or PIK3CA expression at analysis cut-off date (median PFS 7.5 and 8.5 months respectively vs. 8.9 and 9.0 months respectively; p = 0.502 and p = 0.268 respectively). There remained no significant difference in PFS after having adjusted for significant confounders (HR 1.2 and 1.1 respectively; 95% CI 0.7–1.9 and 0.7–1.8 respectively; p = 0.481 and p = 0.730 respectively). The distribution of p95HER2 expression and tumor HER mRNA levels and their association with PFS will be included at the time of presentation.nnOur preliminary findings suggest that PTEN alteration, or PIK3CA mutation may not be predictive of clinical response to lapatinib treatment in HER2+ breast cancer patients. The final PFS results with additional markers will provide more clues regarding their relationship to treatment response.nnCitation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-28.

Abstract P1-14-04: Clinical Outcomes of Brain Metastasis by Lapatinib (L) and Capecitabine (C) in an Open-Label Expanded Access Study among Korean Patients with HER2 Positive Metastatic Breast Cancer

Background: The L-Expanded Access Program (LEAP) was designed to provide access to L/C for HER2+ patients (pts) who previously received anthracycline, taxane, and trastuzumab. In earlier registration trial, CNS metastases occurred in fewer women receiving L/C therapy compared with C alone. Although L therapy has shown efficacy in brain metastasis (BM), it has not been well elucidated in relationship with the outcomes of systemic disease. Patients and methods : Enrollment in LEAP lasted between Jan. 2007 and Apr. 2008 at 6 centers in Korea. Exploratory analysis on clinical outcomes of brain metastasis was performed. Results: Total 187 pts enrolled in LEAP. The median treatment duration of all pts was 19.0 weeks (range, 1.4 — 146.9). The median progression free survival and overall survival (OS) of all pts were 20.0 (95% confidence interval [CI]= 18.6-24.0) and 60.0 (95% CI= 50.3-72.7) weeks, respectively. All patients received prior trastuzumab therapy, and 48.7% received prior capecitabine. Among 58 enrolled pts who had BM diagnosed before start L/C therapy, 55 pts were included for the analysis excluding 3 pts (1-consent withdrawal and 2- Conclusion: Overall survival of patients with brain metastasis who received lapatinib plus capecitabine was prolonged specifically in responders of both brain and extracranial disease. Patients with hormone receptor positive tumors had longer survival compared with those of hormone receptor negative disease. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-14-04.