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Phylloplane yeast genera Pseudozyma and Cryptococcus secrete biodegradable plastic (BP)-degrading enzymes, termed cutinase-like enzymes (CLEs). Although CLEs contain highly conserved catalytic sites, the whole protein exhibits ≤30% amino acid sequence homology with cutinase. In this study, we analyzed whether CLEs exhibit cutinase activity. Seventeen Cryptococcus magnus strains, which degrade BP at 15 °C, were isolated from leaves and identified the DNA sequence of the CLE in one of the strains. Cutin was prepared from tomato leaves and treated with CLEs from 3 Cryptococcus species (C. magnus, Cryptococcus flavus, and Cryptococcus laurentii) and Pseudozyma antarctia (PaE). A typical cutin monomer, 10,16-dihydroxyhexadecanoic acid, was detected in extracts of the reaction solution via gas chromatography–mass spectrometry, showing that cutin was indeed degraded by CLEs. In addition to the aforementioned monomer, separation analysis via thin-layer chromatography detected high-molecular-weight products resulting from the breakdown of cutin by PaE, indicating that PaE acts as an endo-type enzyme.

The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.

The yeast Pseudozyma antarctica secretes a concentrated biodegradable plastic (BP)-degrading enzyme when cultivated with xylose. Treatment with the culture filtrate reduced the puncture strength of commercial BP mulch films. After burying the film in soil, the residual amount of solid film was reduced significantly, and none was recovered after 5 weeks. The dynamics of soil fungal communities were analyzed weekly after burying the film using 18S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiling of soil DNA. In the soil containing enzyme-treated film, the native community essentially recovered within 24 weeks. In comparison, the untreated solid film remained in the soil for 12 weeks and the response of the soil-fungal community was relatively slow; it had not recovered within 24 weeks.

Biodegradable plastics must be sufficiently stable to maintain functionality during use but need to be able to degrade rapidly after use. We previously reported that treatment with an enzyme named PaE, secreted by the basidiomycete yeast Pseudozyma antarctica can speed up this degradation. To facilitate the production of large quantities of PaE, here, we aimed to elucidate the optimal conditions of ethanol treatment for sterilization of the culture supernatant and for concentration and stabilization of PaE. The results showed that Pseudozyma antarctica completely lost its proliferating ability when incubated in ≥20% (v/v) ethanol. When the ethanol concentration was raised to 90% (v/v), PaE formed a precipitate; however, its activity was restored completely when the precipitate was dissolved in water. To reduce ethanol use, PaE was successfully concentrated and recovered by sequential ammonium sulfate precipitation and ethanol precipitation steps. Over 90% of the activity in the original culture supernatant was recovered and the specific activity was increased 3.4-fold. By preparing the enzyme solution at a final concentration of 20% (v/v) ethanol, about 60% of the initial activity was maintained at ambient temperature for over 6 months without growth of microbes. We conclude that ethanol treatment is effective for sterilization, concentration, and stabilization of PaE, and that concentrating PaE by sequential ammonium sulfate precipitation and ethanol precipitation substantially increases the PaE purity and decreases ethanol use.

Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.

The enhancement mode AlGaN/GaN hybrid MOS-HFETs on Si substrates have been demonstrated. The breakdown voltage of over 1.71 kV was achieved by investigating the epitaxial structure. Furthermore, a high integrity SiO2/Al2O3 gate stack has been demonstrated for GaN MOSFETs. The SiO2 film formed on GaN by the MW-PECVD exhibits good properties compared that by the LP-CVD. Then, by incorporating the advantages of both of SiO2 with a high insulating characteristics and Al2O3 with good interface characteristics, the SiO2/Al2O3 gate stack structure has been employed in GaN MOS devices. It is shown that a low interface state density between gate insulator and GaN, a high breakdown field, and a large charge-to-breakdown by applying 3-nm Al2O3 in this structure. The SiO2/Al2O3 gate stack has also been applied to AlGaN/GaN hybrid MOS-HFET and excellent properties with the threshold voltage of 4.2 V and the maximum field-effect mobility of 192 cm2/Vs are shown in the transistor.

Summary Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.

By combining immunohistochemical technique with microassay methods, we analyzed regional energy metabolism in vulnerable and tolerant areas of gerbil brains during evolution of neuronal damage after bilateral common carotid artery occlusion for 10 min with subsequent reperfusion. Four animals were used for each reperfusion period. Based on the information from the immunohistochemical examination, we dissected out vulnerable and tolerant subregions of the hippocampus, cerebral cortex, and thalamus from freeze-dried 20-microm-thick sections, and measured the levels of creatine phosphate (P-Cr), adenine nucleotides, guanine nucleotides, and purine bodies by HPLC, and the levels of glucose, glycogen, and lactate by an enzyme-immobilized column method. There were no significant differences in the levels of metabolites between vulnerable and tolerant subregions of control brains. After reperfusion, both vulnerable and tolerant subregions recovered preischemic metabolic profiles by 2 days. Although the regional differences between vulnerable and tolerant subregions were minimal at each reperfusion period, there were delays in the recovery of P-Cr, ATP, and/or total adenine nucleotides in all vulnerable subregions. A decline of P-Cr, ATP, and GTP levels without change in %ATP, AMP, or purine bodies occurred after reperfusion for 3 days, coinciding with the development of immunohistochemical damage by the immunoreaction for microtubule-associated protein 1A. The results supported the notion that subtle but sustained impairment of energy metabolism caused by mitochondrial dysfunction in the early reperfusion period might trigger delayed neuronal death in vulnerable subregions.

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of 'protein machine'.

The authors compared temporal profiles of Nacetylaspartate (NAA) and the NAA/total creatine ratio with neuronal and astrocytic densities and with tissue atrophy in the hippocampal CA1 sector of gerbils after 5-minute bilateral forebrain ischemia and subsequent reperfusion for up to 6 months. The CA1 sector was dissected from 20-μm lyophilized sections (n = 5) for NAA, phosphocreatine, and creatine assays using high-performance liquid chromatography. Adjacent 10-μm sections were used for immunohistochemical analysis to follow neuronal and astrocytic responses. The NAA concentration was significantly ( P

Protein synthesis is important in the readaptive processes for cultured astrocytes after hypoxia and subsequent reoxygenation. We have identified 72-kDa inducible heat shock protein (HSP72) as a major stress protein in reoxygenated astrocytes. To assess the mechanism for reoxygenation-mediated induction of HSP72, a reporter gene that consists of a human HSP promoter fused to the luciferase gene was transfected into cultured astrocytes. Analysis of cellular energy nucleotides showed an increase of the ADP/ATP ratio after reoxygenation, which synchronized with activation of the HSP promoter. Activation of the HSP promoter was also observed after an addition of iodoacetic acid to hypoxic astrocytes, which reached the maximum when the ADP/ATP ratio reached 50%, but further decline in the energy profile caused inactivation of this promoter. Inhibition of protein synthesis after reoxygenation resulted in temporary restoration of the energy profile and suppression of the DNA binding activity of the heat shock factor. Addition of quercetin greatly decreased the [3H]leucine incorporation in the polysome fraction without any effect on the mature mRNA formation. These data suggest that the energy depletion in reoxygenation triggers induction of HSP72 after reoxygenation, which may act as a pivotal mediator in the stress response of reoxygenated astrocytes by facilitating protein synthesis.

Natural pyrethrins biosynthesized by Chrysanthemum cinerariaefolium exhibit insecticidal activity, thereby acting as a defense system against herbivores. Here, we study the effects of mechanical wo...

By analyzing histological damages and the regional N-acetylaspartate (NAA) level simultaneously, we evaluated the effect of an ∂-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist, YM90K [6-(1 H -imidazol-1-yl)-7-nitro-2,3-(1 H ,4 H )-quinoxalinedione monohydrochloride], in unilateral forebrain ischemia in gerbils. The right common carotid artery was clipped for 5 min under ether anesthesia, and reperfused for 7 days. The frozen brain sections were lyophilized and the hippocampal CA1 area was dissected out for HPLC assay of NAA. An adjacent section was stained with hematoxylin-eosin for counting survived neurons per 1 mm pyramidal layer of the hippocampal CA1 area. Postischemic administration of YM90K at 20 mg/kg and 25 mg/kg attenuated the decrease of both the number of survived neurons and the NAA level on the ischemic side in a dose-dependent manner. A significant linear correlation was observed between the NAA level and the number of intact neurons. These results indicated that the NAA level could be used as an index of neuroprotective effects of pharmacological agents in global cerebral ischemia.

We investigated the correlation between N-acetylaspartate (NAA) level and neuronal density in the hippocampal CA1 region of the brain after occlusion of both common carotid arteries for 5 minutes and reperfusion for 3 hours to 4 weeks in gerbils with and without ischemic preconditioning (tolerance). Animals were divided into four groups--the sham operated group, the nonpreconditioning (non-p) group, the single-preconditioning (single-p) group with 2-minute ischemia once 2 days before 5-minute ischemia, and the double-preconditioning (double-p) group with 2-minute ischemia twice 2 days before 5-minute ischemia (n = 6 for each group). The CA1 region was dissected out from freeze-dried sections for high-performance liquid chromatographic assay of NAA, and adjacent sections were stained with cresyl violet for measurement of the neuronal density. Both NAA (pmol/microg dry weight) and the neuronal density (cells/mm) decreased in the non-p group after 3 days (NAA = 24.0 +/- 3.0; neuronal density = 65 +/- 38 cells/mm) and 7 days (NAA = 17.9 +/- 2.5; neuronal density = 20 +/- 15 cells/mm) and in the single-p group after 7 days (26.4 +/- 3.0, 106 +/- 30) compared with the control group (NAA = 32.9 +/- 3.0; neuronal density = 203 +/- 9 cells/mm). There was no decrease in the double-p group. The NAA level and the neuronal density showed a good linear correlation. The regional NAA level may be used as an index of neuronal viability.

Background and Purpose To explore the physiological mechanism of ischemic tolerance, we studied intracerebral oxygenation states noninvasively using near-infrared spectroscopy after bilateral common carotid artery occlusion (BCO) in gerbils with and without ischemic pretreatment. Methods Under ether anesthesia, gerbils with sham operation (S group, n=8) and those with pretreatment consisting of BCO for 2 minutes, twice at 3 days and 2 days earlier (T group, n=8), were again subjected to BCO for 5 minutes. Changes in oxyhemoglobin (HbO 2 ), deoxyhemoglobin (Hb), and total hemoglobin (HbT) as well as reduction in cytochrome oxidase (cyt. aa 3 ) were calculated from the absorbance changes of the light transmitted through the brain. Seven days after the ischemic study, immunohistochemical examination was performed with an antiserum against microtubule-associated proteins. Results In both groups, the increase of Hb and decrease of HbO 2 and HbT proceeded rapidly after BCO, and the maximal deoxygenation of hemoglobin occurred within 2.5 minutes. Reduction of cyt. aa 3 also ensued rapidly and reached the maximal reduction within 3 minutes in both groups. In the T group, however, both deoxygenation of hemoglobin and reduction of cyt. aa 3 progressed more slowly than in the S group. The time (seconds) necessary for a maximal change for cyt. aa 3 was significantly longer in the T group (203.8±34.0 [mean±SD]; P P aa 3 (23.9±5.7 and 11.6±4.3; P Conclusions The present study indicated that mild ischemic stress can induce improvement in oxygen metabolism during subsequent ischemia, which might be causally related to the phenomenon known as “ischemic tolerance,” in which a protective effect toward ischemic/postischemic injury is induced by earlier mild ischemic pretreatment.

In view of the potential relationship between cerebral ischemia and zinc status, this study was designed to examine whether dietary zinc-deficiency would affect the outcome of focal cerebral ischemia in rats. Forty-five male Sprague-Dowley rats were randomly divided into five groups: (1) eight rats received a semi-purified zinc-deficient diet containing less than 0.38mg/100g of zinc for 1 week (ZD-1w); (2) eleven rats received the same diet as described as above for 2 weeks (ZD-2w); (3) ten rats, named pair-fed control group (PF-2w), received a zinc-supplemented diet and matched by the amount of diet equal to that consumed by ZD-2w group for 2 weeks; (4) ad libitum-fed control group received the same diet as taken by PF-2w animals for 1 week (AF-1w, n=6) and (5) the remaining 10 rats were named as no-fed (NF) group, in which the rats were directly subjected to cerebral ischemia without entering the feeding process. After experiencing the dieting stage for 1 or 2 weeks, or without (NF group), respectively, the animals were subjected to permanent occlusion of the middle cerebral artery (MCA). Plasma zinc concentration and the cerebral infarct volume were determined at 24 hours of ischemia. The final level of zinc in NF and AF-1w rats was 139±5 and 127±6 μg/100 ml, respectively. The zinc-deficient diet reduced the concentration by 52% in ZD-1w and by 64% in ZD-2w group. The increment in infarct size in ZD-1w group was inconspicuous. However, treatment with zinc-deficient diet for 2 weeks increased the total infarct volume by 118%, 88%, 58% and 47% as compared with NF, AF-1w, PF-2w and ZD-1w rats, respectively. Especially, the cortical infarct volume (206±35 mm 3 ) in ZD-2w group was 98–227% larger than those in the other groups (p

Tobacco genes regulated during the hypersensitive response (HR) to infection by tobacco mosaic viruses were screened by differential display and one particular clone with a rapid expression profile was selected and further characterized. The obtained cDNA encoded a polypeptide of 205 amino acids with an apparent molecular mass of 23 kDa and a pI of 10.37 and production of a GFP fusion protein revealed localization in the cytoplasm. Transcripts of the clone were strongly induced in leaves 3 h after HR onset, and 10 min after mechanical wounding, as well as by treatment with jasmonic and salicylic acids. The induction profile satisfies the definition of pathogenesis-related (PR), and the protein was designated as NtPRp23. Database searches indicated the presence of similar proteins in Arabidopsis and rice, but with functions yet to be assigned. Our results point to the existence of a novel PR protein family in tobacco plants.

High integrity SiO2/Al2O3 gate stack has been demonstrated for GaN metal-oxide-semiconductor (MOS) transistors. The SiO2 film formed on GaN by the microwave-excited plasma enhanced chemical vapor deposition (MW-PECVD) exhibits good properties compared that by the LP (Low Pressure)-CVD. Then, by incorporating the advantages of both of SiO2 with a high insulating and Al2O3 with good interface characteristics, the SiO2/Al2O3 gate stack structure has been employed in GaN MOS devices. The structure shows a low interface state density between gate insulator and GaN, a high breakdown field, and a large charge-to-breakdown by applying 3-nm Al2O3. The SiO2/Al2O3 gate stack has also been applied to AlGaN/GaN hybrid MOS heterojunction field-effect transistor (HFET) and the HFET shows excellent properties with the threshold voltage of 4.2 V and the maximum field-effect mobility of 192 cm2/Vs.

Glutamate triggers neuronal degeneration after ischemia-reperfusion in the brain. However, the details of intracellular signal transduction that propagates cell death remain unknown. The present work investigated whether protein tyrosine phosphorylation mediates neuronal death in the ischemic brain. Transient forebrain ischemia for 5-10 min in Mongolian gerbils or intoxication with the glutamate analogue kainic acid (12 mg/kg) in Sprague-Dawley rats caused neuronal death selectively in the hippocampus 2-4 days or 1 day later, respectively. Under these conditions, 160-, 115-, 105-, 92-, and 85-kDa proteins showed a significant increase in tyrosyl residue phosphorylation selectively in the hippocampus 3-12 h after ischemia or 4-8 h after kainic acid-induced seizures. Tyrosine kinases, including pp60c-src, were activated without a change of tyrosine phosphatases. Administration of radicicol, a selective inhibitor of tyrosine kinases, attenuated stimulation of tyrosine phosphorylation and hippocampal degeneration after ischemia or kainic acid injection. The results suggest that protein tyrosine phosphorylation might propagate delayed neuronal death in the mature hippocampus through glutamate overload after ischemia-reperfusion.

The lithium rare earth silicates of nominal composition “LiLnSiO 4 ” (Ln = La, Nd, Sm, Eu, Gd, Dy) have been synthesized. The lithium ion conductivity of their sintered samples was found to consist of two contributions; one is the grain boundary composed possibly of lithium silicate amorphous phases and the other the bulk phase with an apatite structure. Although the conductivity of the bulk phase was smaller than that of the grain boundary, it was apparently increased by several orders of magnitude than that of stoichiometric apatites of Li x Ln 10 − x Si 6 O 24 O 3 − x with x = 2 and x = 3. From the structure refinement of the bulk phase, this enhancement in the conductivity of the apatite phase in LiLnSiO 4 was reasonably interpreted by the model where lithium ions and vacancies were both partially introduced to the 4 f site of the apatite structure.

Next generation semiconductor device technology node logic and advanced memory fabrication etch [1] deposition [2] and functionalization [3,4] unit processes require management of process chamber species’ nature, flux and energy at all facets of a substrate and feature being fabricated. Management of profile (conformality), selectivity, deposition or etch rate, uniformity, damage and/or sub-surface and film “quality” needs via continuous or quasi-continuous processes is difficult as process chemistries and plasma source characteristics drive trade-offs innate to their combination. Fortified by more than two decades of progress in modelling and simulation, concurrent process and equipment development has overcome these difficulties. It is evident now to meet trade-off born difficulties in the future that self-limiting process technology will need to be used. In this case, chemistry control may mean parsing individual process steps into adsorption and desorption components [5]. Segregating processes include rapid cyclic etch and atomic layer deposition (ALD). Other examples of self-limiting processes include radical driven plasma doping. This presentation will discuss each of these processes in the context of their needs and how simulation drives their development. Simulation models discussed are based on an integrated approach (chamber through feature) with fluxes to surfaces from plasma source simulations (e.g., HPEM [6]) mated to feature scale simulations such as MCFPM. [7] Atomic layer etching for front end applications will be described that use this suite of simulation models. [8] While atomic layer deposition is more mature than it’s etch counterpart, plasma assisted ALD comes with mechanistic complexity both in the plasma and at surface. [9] Fortunately, quantum chemistry methods, [10] surface and plasma chemistry models can be linked to equipment simulations for concurrent process design. Three dimensional devices require source drain engineering to being about conformal electrical properties. An example is fin extension doping. Radical driven plasma doping can be conformal because of the self-passivating nature of a saturation layer created all around the surface of the fin during the doping process. Functionalizing topographic structures such as silicon, Ge or III-V fins sets severe constraints on the plasma itself. Fins must not erode but sufficient ion energy is required to disorder a single surface layer so that dopants may be infused. Plasma equipment simulations show that high microwave power and high pressure operation are sufficient to generate sufficient dopant radicals with a sufficient non-damaging flux of light ions to the surface to meet functionalization requirements. [11] The properties of the surface layer are very important. Using arsenic doping of silicon as an example, first principles calculations of ternary dopant silicon oxygen system were used to explore the surface film properties. [12] The calculations show that trade-offs between strain relaxation of the amorphous and the evolution of penalty energy as arsenic is introduced to the amorphous silica system creates a preferred stoichiometry film. This is one ingredient to maintaining conformality in the presence of different flux compositions all around a feature. The presentation will close with a look forward at how the new norm in modeling and simulation will involve addition of first-principles (quantum chemistry through device) to the integrated source through feature approach of the last two decades. [1] C. T. Carver et al., ECS J. Sol. St. Sci. Tech., 4 N5005-N5009 (2015) [2] S. M. George, Chem. Rev., 110, 111 (2010) [3] K. J. Kuhn, IEEE Trans. Elec. Dev., 59, 1813 (2012) [4] T. Tatsumi and J. Lee, Jpn. J. Appl. Phys., 53, 06JE06 (2014) [5] K. Ono and M. Tuda, Thin Solid Films, 374, 208 (2000) [6] M. Wang and M. J. Kushner, J. Appl. Phys 107, 023308 (2010) [7] A. Agarwal and M. J. Kushner, J. Vac. Sci. Technol. A 26, 498 (2008) [8] A. Ranjan et al., Proc. SPIE 9428, Advanced Etch Technology for Nanopatterning IV, 94280O (2015) [9] S. King, J. Vac. Sci. Technol. A, 29, 041501(2011) [10] J. Yoshikawa, AVS 60th Annual Symposium, Paper TF+PS-ThM12(2013) [11] H. Ueda et al., J. Appl. Phys., 115, 214904 (2014) [12] P. Ventzek et al., Appl. Phys. Lett., 105, 262102 (2014)

Microwave RLSA™ plasma doping technology has enabled conformal doping of non-planar semiconductor device structures. An important attribute of RLSA™ plasma doping is that it does not impart physical damage during processing. In this work, carrier activation measurements for AsH3 based plasma doping into silicon (Si) and germanium (Ge) using rapid thermal annealing are presented. The highest carrier concentrations are 3.6 × 1020 and 4.3 × 1018 cm−3 for Si and Ge, respectively. Secondary ion mass spectrometry depth profiles of arsenic in Ge show that intrinsic dopant diffusion for plasma doping followed by post activation anneal is much slower than for conventional ion implantation. This is indicative of an absence of defects. The comparison is based on a comparison of diffusion times at identical annealing temperatures. The absence of defects, like those generated in conventional ion implantation, in RLSA™ based doping processes makes RLSA™ doping technology useful for damage free conformal doping of topographic structures.

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.

Plants emit volatile organic compounds (VOCs) as a means to warn other plants of impending danger. Nearby plants exposed to the induced VOCs prepare their own defense weapons in response. Accumulated data supports this assertion, yet much of the evidence has been obtained in laboratories under artificial conditions where, for example, a single VOC might be applied at a concentration that plants do not actually experience in nature. Experiments conducted outdoors suggest that communication occurs only within a limited distance from the damaged plants. Thus, the question remains as to whether VOCs work as a single component or a specific blend, and at which concentrations VOCs elicit insect and pathogen defenses in undamaged plants. We discuss these issues based on available literature and our recent work, and propose future directions in this field.

The chemical lithium intercalation of a layered perovskite LiLaNb2O7 compound has been performed by using n-butyllithium. The content of inserted lithium ions was found to be ca. 1 mol per unit formula. The crystal structure of the intercalated compound was determined by the powder X-ray diffraction pattern. An unusual contraction of the cell volume inherent in the intercalation reaction was observed.

Plants emit specific blends of volatile organic compounds (VOCs) in response to mechanical wounding. Such induced VOCs have been shown to mediate in plant and interplant communication, yet little is known about the time- and dose-response relationships in VOC-mediated communications. Here, we employed young seedlings of Chrysanthemum cinerariaefolium to examine the effects of volatiles emitted by artificially damaged seedlings on the biosynthesis of the natural insecticides pyrethrins in intact conspecific plants. Wounded leaves emitted (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate and (E)-β-farnesene as dominant wound-induced VOCs. Exposing intact seedlings to a mixture of these VOCs at concentrations mimicking those emitted from wounded seedlings, as well as placing the intact seedlings next to the wounded seedlings, resulted in enhanced pyrethrin contents in the intact seedlings. Thus we quantified mRNA transcripts of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), chrysanthemyl diphosphate synthase (CPPase), 13-lipoxygenase (13-LOX) and allene oxide synthase (AOS) genes in intact seedlings exposed to the VOC mixture to show that DXS and 13-LOX gene expression reached a maximum at 3 h, whereas CPPase and AOS reached it at 6 h. Interestingly, both increasing and decreasing the VOC mixture concentrations from those observed on injury reduced the expression of DXS, CPPase and AOS genes to the control level. Also, separating the VOC mixture into individual components eliminated the ability to enhance the expression of all the biosynthetic genes examined. This is the first study showing that the wound-induced VOCs function as a blend to control the biosynthesis of second metabolites at specific concentrations.

Brief periods of non-lethal cerebral ischemia can induce resistance against subsequent lethal ischemia. In this study, asymptomatic gerbils after unilateral carotid artery ligation were subjected to 5 min of forebrain ischemia. The prolonged but mild hypoperfusion, by carotid occlusion, induced susceptibility at 1 day and tolerance at 30 days to lethal ischemia in the hippocampal neurons. The neuroprotective effect correlated well with induction of heat shock protein 72 in the hippocampal neurons. These results suggested that neuronal cells possess a cellular response to sublethal hypoperfusion and can survive forthcoming ischemic stress.

LiYSiO4 was synthesized by sol-gel method and its crystal structure was determined by the Rietveld analysis. The structure was found to be an olivine-type structure with orthorhombic Pnma space group. The thin film deposited on an quartz substrate could be prepared by a dip-coating method using sol solutions consisting of lithium i-propoxides, silicon tetraethoxide and yttrium acetate.

Treatment of 2-propynyl alcohols with several nucleophiles in the presence of a catalytic amount of a rhenium complex, [ReBr(CO)3(thf)]2, gave coupling products via dehydration. In these reactions,...

Isomerization between two isomers of 1,2-disubstituted 3-aminoindenes occurs via the rearrangement of indene frameworks. In contrast to previous rearrangements of indene derivatives, which occur under high-temperature conditions or the irradiation of light, this rearrangement proceeds at room temperature without UV light irradiation. An amino group at the 3-position plays an important role to accelerate the rearrangement under mild conditions.

Isomerization between two isomers of 1,2-disubstituted 3-aminoindenes occurs via the rearrangement of indene frameworks. In contrast to previous rearrangements of indene derivatives, which occur under high-temperature conditions or the irradiation of light, this rearrangement proceeds at room temperature without UV light irradiation. An amino group at the 3-position plays an important role to accelerate the rearrangement under mild conditions.

Vasoconstriction is known to occur in cerebral arterioles during ischemia and considered to be distinct from vasospasm seen after subarachnoid hemorrhage. To elucidate the mechanism and functional significance underlying ischemic vasoconstriction, we investigated the relationship between arteriolar constriction and tissue energy metabolism during bilateral common carotid artery occlusion in gerbils. Using video microscopy and microspectroscopy, the arteriolar caliber, the total hemoglobin (Hb) content, and the redox state of cytochrome oxidase (cyt.aa3) were monitored in the cerebral cortex in vivo. After in situ freezing of the brain, adenine nucleotides, creatine phosphate (P-Cr), and lactate levels were analyzed using high-performance liquid chromatography in vitro. Tissue damage was also assessed immunohistochemically using antibodies against microtubule-associated proteins. There was a slight reduction of the diameter of pial arterioles during the initial 1 min of ischemia. A rapid decline of total Hb and reduction of cyt.aa3 were observed with rapid decreases of P-Cr and ATP in the cortical tissue during the initial 0.5 min, but all of them showed tendencies to return toward preischemic levels at 0.5-1 min. Beyond 1.5 min, extensive vasoconstriction occurred together with further decline of total Hb, reduction of cyt.aa3, and decreases of ATP and P-Cr. Neuronal damage developed in the cerebral cortex immunohistochemically beyond 3 min. The present investigation demonstrated two phases of vasoconstriction with the possibilities that the immediate vasoconstriction likely contributed to transient improvement of cortical oxygen/energy metabolism, and the second extensive vasoconstriction was an index of tissue energy failure and imminent neuronal damage.

We demonstrate stable arsenic-silicon-oxide film formation during plasma doping of arsenic into non-planar silicon surfaces through investigation of the nature and stability of the ternary oxide using first principles calculations with experimental validations. It is found that arsenic can be co-mingled with silicon and oxygen, while the ternary oxide exhibits the minimum energy phase at x ≈ 0.3 in AsxSi1−xO2−0.5x. Our calculations also predict that the arsenic-silicon-oxide alloy may undergo separation into As-O, Si-rich As-Si-O, and Si-O phases depending on the composition ratio, consistent with experimental observations. This work highlights the importance of the solid-state chemistry for controlled plasma doping.

Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.

We investigated changes in regional N-acetyl-aspartate (NAA) levels in the vulnerable CA1 and resistant CA3 areas of the hippocampus after transient forebrain ischemia in gerbils. Under light ether anesthesia, bilateral common carotid arteries of adult male Mongolian gerbils (60–80 g) were occluded for 5 min and reperfused for 7 days. Brains from experimental and control gerbils (n = 4 each) were frozen in situ, and frozen sections (20 μm) were prepared using cryostat (-20°C). After overnight lyophilization, the CA1 and CA3 areas were dissected out separately, weighed (50–200 μg), and the supernatant of the perchloric acid extract was used for assay of NAA using HPLC. Adjacent 10 μm-thick sections were used for immunohistochemical analysis using antiserum against microtubuleassociated protein I and II. The preischemic NAA levels were not significantly different between CA1 and CA3 areas. After transient ischemia, a significant (P < 0.01) decrease in the NAA level was observed in the CA1 area, but not in the CA3 area of the hippocampus. Immunohistochemical ischemic damage evolved only in the CA1 area. Thus, the decrease of the regional NAA level was associated with development of immunohistochemical neuronal damage.