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To clarify the factors affecting 137Cs transfer from soil to brown rice in comparison with 133Cs transfer and also to determine the key parameters of the solid-liquid partition coefficient (K d), c...

Land use shows the interaction between human and nature, and its impacts especially on runoff have received global attention and required more research studies. However, watershed database is generally available only for very large scales and not yet adapted to small scales. This paper presents a methodology for watershed delineation and potential influence assessment of runoff on the small watersheds in Japan based on the locations of the official observatory point data. Flow direction, flow accumulation, snap pour point and watershed recognition were analyzed by using the D8 algorithm and 10 m × 10 m DEM data of Japan. Totally 3831 watersheds were delineated by defining snapping pour point distance of 50 m as the input value, and the nation-wide watershed database was established. According to the suggestion of Notification No. 521 from the Ministry of Land, Infrastructure and Transport of Japan and land use/land cover data from JAXA satellite Alos-2, comprehensive runoff coefficient (CRC) was calculated by the weighted average method and divided into 10 levels by the equal interval method using ArcGIS. The obtained watershed boundary lines were almost identical to the ridge lines in the mountain area which accounts for more than half of Japan. The CRC values in the biggest cities such as Tokyo and Osaka were the highest, indicating these cities would receive the highest impacts of runoff. These results can provide the technical support for the decision-making on watershed management. The creation of small-scale watershed database would provide basic materials for the subsequent research such as flood prevention, water shortage and biogeochemical cycle of the sustainable regional development.

To examine the effects of phenolic acids, which are generated by the decomposition of cell walls in plant residues, and other constituents on the stability of soil aggregates, phenolic acids and carbohydrates were mixed into three different types of soil. After a 1-month incubation, the plot containing soil mixed with phenolic acids showed the greatest mean weight diameter of all the soils. In the treated soils, before incubation, the decline of saturated water permeability during continuous water percolation was mitigated in the plot containing soil mixed with phenolic acids compared with that in the other plots. Soil aggregates were synthesized with the addition of phenolic acids and carbohydrates using two methods (mixing and surface brushing) and were incubated for 153 days. The aggregate stability was greatest in the plots surface-brushed with phenolic acids for Andosol and gray lowland soil, whereas the aggregate stability was most stable in the plots mixed with phenolic acids for yellow soi...

Rice absorbs radiocesium dissolved in soil solution. However, quantitative determination of dissolved radiocesium is difficult because large volume of water should be collected from paddy soil. We investigated whether a sheet capable of effectively trapping cesium (zinc-substituted Prussian blue (ZnPB) sheet) can be used to evaluate the level of dissolved 137Cs to be absorbed by rice. A new device equipped with ZnPB sheet was invented to trap only radiocesium dissolved in soil solution, which is passed through a membrane filter covering the ZnPB sheet. A rice pot-culture experiment using three paddy soils with different 137Cs concentrations and physico-chemical properties collected from Fukushima Prefecture was conducted under different fertilization or rice-planting treatments. The results showed that 137Cs concentration in brown rice was positively correlated with the 137Cs concentration in soil solution with a contribution ratio of regression R2 of 0.91 and a p value less than 0.01, and also with the 137Cs-trapping rate by ZnPB sheet (amount of 137Cs trapped by ZnPB sheet per unit area of sheet and unit time) buried in soil with a R2 value of 0.89 and a p value less than 0.01. Moreover, field-cropping experiments conducted at the three paddy fields under conventional agricultural practices showed positive correlation between the 137Cs concentration in brown rice and 137Cs-trapping rate by ZnPB sheet. Both the pot and field experiments showed that the 2 weeks of the early stage of rice growth is appropriate as the ZnPB buried period because K additive fertilization is still in time to restrict 137Cs uptake by rice plant, and because the highest 137Cs-trapping rates of the ZnPB plate as compared with the following periods and high R2 values of those regression curves. . The proposed method might be used to roughly evaluate the vulnerability of soil to 137Cs transfer to rice and to identify the area in which effective countermeasure should be applied intensively.

Hiso River watershed (HRW) is within a radiocesium contaminated area caused by the disaster in Fukushima Daiichi nuclear power plant (FDNPP). The FDNPP accident resulted in enormous changes in land use in HRW. It's urgently needed to evaluate impacts of climate and land-use changes on hydrological process, which further control pollutants transport in HRW. A combination method of Statistical DownScaling Model (SDSM) and Soil and Water Assessment Tool (SWAT) was applied to generate future climatic and hydrologic variables. Future climate data was obtained from three Representative Concentration Pathway (RCP2.6, 4.5 and 8.5) scenarios of a single General Circulation Models in three future periods of 2030s, 2060s and 2090s (2010–2039, 2040–2069, 2070–2099), with a baseline period (1980–2009). According to land-use change in HRW during 2013–2017, three land-use change scenarios under the three future climate scenarios were established. Results suggested that SDSM showed good capabilities in capturing daily maximum/minimum temperature and precipitation. The SWAT model presented good performances in simulating monthly and yearly streamflow. Results also suggested projected higher temperatures and lower rainfall led to decreased annual water yield and evapotranspiration (ET). The annual water yield and ET decreased in most seasons while had a slight increase in spring. RCP8.5 scenario always generated larger magnitudes for climatic variables and water balance components compared with other climate scenarios. Land-use changes had strong impact on surface runoff and groundwater flow. These results emphasize the necessity of considering impacts of climate and land-use changes on hydrological processes when making decontamination policy in radiocesium contaminated areas.

The aim of this study was to evaluate the impact of land use on nitrate nitrogen (NO3-N) in shallow groundwater (G-N) and total nitrogen (N) in river water (R-N). The study area consisted of 26 watersheds (1342 km2) covering 72% of Kagawa Prefecture in Japan. We estimated G-N specific concentrations, which showed the magnitude of the upland fields, paddy fields, forests and urban land-use contributions to watershed-mean G-N. G-N specific concentrations were gained as partial regression coefficients using a multiple regression analysis of the watershed-mean G-N concentrations and the land-use ratios in each of the 26 watersheds. The results showed that the G-N specific concentration, which was gained as the partial regression coefficient for the multiple regression analysis, was 15.2 mg L−1, 10.3 mg L−1, 2.3 mg L−1 and 2.5 mg L−1 for the upland fields, paddy fields, forests and urban land-use types, respectively. R-N pollution load runoff to the river mouth was calculated by multiplying R-N specifi...

To contribute to the prediction of rainfall-related disasters, specific concentration factors that indicate the suspended solid (SS), total nitrogen (TN), and total phosphorus (TP) load intensities to river water for each land-use type were calculated using a simple watershed land-use model across Japan by applying the following multiple regression equation, according to the land-use ratios and published SS, TN and TP data. C=i=14aixi C: SS, TN and TP concentrations (mg L-1); ai: SS, TN and TP specific concentration factor for land use i; xi: ratio of land use i; land use: 1 paddy fields, 2 upland fields, 3 forests, 4 urban areas. The land-use ratios for watersheds, whose lower ends were observation points of river water quality, were determined by the GIS technique using a published database of DEM and LULC mesh data. The SS specific concentration factor was 15.4 (from a 95% lower limit value of 12.0 to a 95% upper limit value of 18.8), 11.5 (7.4 to 15.6), 3.9 (2.6 to 5.1), and 11.2 (9.2 to 13.2) for paddy fields, upland fields, forests and urban areas, respectively (n=5103). The TN specific concentration factor was 1.67 (from a 95% lower limit value of 1.34 to a 95% upper limit value of 2.01), 4.08 (3.64 to 4.51), 0.76 (0.67 to 0.90), and 3.57 (3.38 to 3.76) for paddy fields, upland fields, forests and urban areas, respectively (n=3256). The TP specific concentration factor was 0.146 (from a 95% lower limit value of 0.119 to a 95% upper limit value of 0.172), 0.172 (0.138 to 0.206), 0.044 (0.033 to 0.055), and 0.267 (0.253 to 0.282) for paddy fields, upland fields, forests and urban areas, respectively (n=3256). These specific concentration factors had regional tendencies, such as suburban or rural, intensive or extensive agriculture, and so on.

The dual isotope approach using the stable isotope ratios of nitrate nitrogen (δ15NNO3) and oxygen (δ18ONO3) is a strong tool for identifying the history of nitrate in various environments. Basically, a rapid procedure for determining δ15NNO3 and δ18ONO3 values is required to analyze many more samples quickly and thus save on the operational costs of isotope-ratio mass spectrometry (IRMS). We developed a new rapid procedure to save time by pre-treating consecutive samples of nitrous oxide microbially converted from nitrate before IRMS determination. By controlling two six-port valves of the pre-treatment system separately, IRMS determination of the current sample and backflush during the next sample pre-treatment period could be conducted simultaneously. A set of 89 samples was analyzed precisely during a 25-h continuous run (17 min per sample), giving the fastest reported processing time, and simultaneously reducing liquid nitrogen and carrier helium gas consumption by 35%. Application of the procedure to an irrigated rice paddy watershed suggested that nitrate concentrations in river waters decreased in a downstream direction, mainly because of the mixing of nitrate from different sources, without distinct evidence of denitrification. Our procedure should help with more detailed studies of nitrate formation processes in watersheds.

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca2+ elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro274, which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases.

The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27(Kip1) peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27(Kip1) and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G(1) arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27(Kip1) peptide could be developed as an anti-cancer drug against prostate cancer.

Risk assessment and design of remediation methods at soil sites polluted with gaseous phase contaminant require an accurate description of soil-gas diffusion coefficient (Dp) which is typically governed by the variations in soil air-filled porosity (va). For undisturbed volcanic ash soils, recent studies have shown that a linear Dp(va) model, taking into account inactive air-filled pore space (threshold soil-air content, va, th), captured the Dp data across the total soil moisture range from wet to completely dry conditions. In this study, we developed a simple, easy to apply, and still accurate linear Dp(va) model for undisturbed volcanic ash soils. The model slope C and intercept (interpreted as va, th) were derived using the classical Buckingham (1904) Dp(va) power-law model, vaX, at two soil-water matric potentials of pF 2 (near field capacity condition) and pF 4.1 (near wilting point condition), and assuming the same value for the Buckingham exponent (X=2.3) in agreement with measured data. This linear Dp(va) prediction model performed better than the traditionally-used non-linear Dp(va) models, especially at dry soil conditions, when tested against several independent data sets from literature. Model parameter sensitivity analysis on soil compaction effects showed that a decrease in slope C and va, th due to uniaxial reduction of air-filled pore space in between aggregates markedly affects the magnitude of soil-gas diffusivity. We recommend the new Dp(va) model using only the soil-air contents at two soil-water matric potential conditions (field capacity and wilting point) for a rapid assessment of the entire Dp-va function.

The soil gas and solute diffusion coefficients and their dependency on soil total porosity (Φ), fluid-phase (air or water) contents, and pore-size distribution largely control chemical release, transport, and fate in soil. The diffusion coefficients hereby play a key role in both local and global environmental issues including spreading, biodegradation and volatilization of hazardous chemicals at polluted soil sites, and soil uptake, production, and emission of greenhouse gases. In a series of papers, we present new advances in describing and predicting the gas and solute diffusion coefficients in variably saturated porous media, carefully distinguishing between repacked and undisturbed media. Also, we establish direct links between gas and solute diffusivity and pore-size distribution, with further links to pore continuity and tortuosity. In this first paper, a porosity correction term is added to a recently presented model for predicting gas diffusivity in repacked soil. The obtained POrosity-Enhanced (POE) model assumes that increased Φ creates additional interconnectivity between air-filled pores. The POE model is tested against data for 18 repacked soils ranging from 0 to 54% clay, including new data measured in this study for both noncompacted and compacted, high-porosity soils. The POE model accurately predicts gas diffusivity across a wide Φ range up to 0.75 m3 m -3 , whereas the original model is accurate only for Φ up to 0.55 m 3 m -3 . A unifying, two-parameter function for gaseous phase pore continuity (f g ) is suggested. The f g function illustrates developments in gas diffusivity models during the last century, including assumptions behind the increasingly precise prediction models for repacked soil. Last, the POE model is coupled with the widely used van Genuchten (vG) soil-water characteristic model, hereby establishing an accurate and predictive link between soil gas diffusivity and pore-size distribution. The closed-form POE-vG gas diffusivity model is highly useful to evaluate effects of pore-size distribution and soil type on gas diffusivity and gas transport in repacked soil systems.

Accurate description of the soil-gas diffusion coefficient (Dp) as a function of air-filled (e) and total (Φ) porosities is required for studies of gas transport and fate processes. After presenting predictive models for Dp in repacked and undisturbed soils (Part I and II), this third paper takes a more descriptive approach allowing for the inclusion of inactive air-filled pore space, e in . Three model-based interpretations of e in are presented: (1) a simple power-law model (labeled Millington-Call) with the exponent (V) taken from Millington (1959; Science 130:100-102), and expanded with a constant e in term (= 0.1 m 3 m -3 ), (2) a model (SOLA) based on analogy with solute diffusion and assuming a linear increase in pore continuity from zero at the threshold air-filled porosity where gas diffusion ceases (e th ) to a maximum at e = Φ, (3) a power-law model (VIPS) assuming variable e in that linearly decreases from a maximum at e = e th to zero at e = Φ. Assuming e th = 0.1 m 3 m -3 , all three models satisfactorily predicted Dp in 18 repacked soils. The difference between the three models is mainly pronounced for higher-Φ soils, and each model has its own advantage. The SOLA model together with similar models for solute diffusivity allows a direct comparison of pore continuity in the soil gaseous and liquid phases, suggesting large differences in tortuosity and inactive fluid-phase between the two phases. The low-parameter Millington-Call model could account for variability in measured Dp along a field transect (Yolo, California) by varying e in with ±0.03 m 3 m -3 3 and is applicable for stochastic gas transport simulations at field scale. The mathematically flexible VIPS model highly accurately fitted D P (e) data for undisturbed soil, illustrating the large possible variations in e th and V. The VIPS model is coupled with the van Genuchten (vG) soil-water characteristic model, yielding a closed-form expression for Dp as a function of soil-water matric potential. The VIPS-vG model is useful to illustrate the combined effects of pore size distribution and inactive pore space on soil-gas diffusivity.

The soil gas diffusion coefficient (D P ) and its dependency on air-filled porosity (e) govern most gas diffusion-reaction processes in soil. Accurate D P (e) prediction models for undisturbed soils are needed in vadose zone transport and fate models. The objective of this paper was to develop a D P (e) model with lower input parameter requirement and similar prediction accuracy as recent soil-type dependent models. Combining three gas diffusivity models: (i) a general power-law D P (e) model, (ii) the classical Buckingham (1904) model for D P at air saturation, and (iii) a recent macroporosity dependent model for D P at -100 cm H 2 O of soil-water metric potential (ψ), yielded a single equation to predict D P as a function of the actual e, the total porosity (Φ), and the macroporosity (e 100 ; defined as the air-filled porosity at ψ = -100 cm H 2 O). The new model, termed the three-porosity model (TPM), requires only one point (at -100 cm H 2 O) on the soil-water characteristic curve (SWC), compared with recent D P (e) models that require knowledge of the entire SWC. The D P (e) was measured at different ψ on undisturbed soil samples from dark-red Latosols (Brazil) and Yellow soils (Japan), representing different tillage intensities. The TPM and five other D P (e) models were tested against the new data (17 sods) and data from the literature for additional 43 undisturbed soils. The new TAM performed equally well (root mean square error [RMSE] in relative gas diffusivity

Soil-water-characteristic-dependent (SWC-dependent) models to predict the gas diffusion coefficient. D r , in undisturbed soil have only been tested within limited ranges of pore-size distribution and total porosity. Andisols (volcanic ash soils) exhibit unusually high porosities and water retention properties. The Campbell SWC model and two Campbell SWC-based models for predicting Dp in undisturbed soil were tested against SWC and Dp data for 18 Andisols and four Gray-lowland (paddy field) soils from Japan. The Campbell model accurately described SWC data for all 22 soils within the matric potential range from -10 to -15 000 cm H 2 O. The SWC-dependent Buckingham-Burdine-Campbell (BBC) gas diffusivity model predicted Dp data well within the same matric potential range for the 18 Andisols. The BBC model showed a minor but systematic underprediction of Dp for three out of the four Gray-lowland soils, likely due to a blocky soil structure with internal fissures. A recent Dp model that also takes into account macroporosity performed nearly as well as the BBC model. However, Dp in the macropore region (air-filled pores >30 μm) was consistently underpredicted, likely due to high continuity of the macropore system in both Andisols and Gray-lowland soils. In agreement with previous model tests for 21 European soils (representing lower porosities and water retention properties), both SWC-dependent D p models gave better predictions for the 22 Japanese soils than soil-type independent models. Combining Dp and SWC data, a so-called gas diffusion fingerprint (GDF) plot to describe soil aeration potential is proposed.

Soil air permeability (k a ) governs convective air and gas transport in soil. The increased use of soil venting systems during vadose zone remediation at polluted soil sites has created a renewed interest in k a and its dependency on soil type and soil air-filled porosity (e). Predictive k a (e) models have only been tested within limited ranges of pore-size distribution and total porosity. Andisols (volcanic ash soils) exhibit unusually high porosities and water retention properties. In this study, measurements of k a (e) on 16 undisturbed Andisols from three locations in Japan were carried out in the soil matric potential interval from -10 cm H 2 O (near water saturation) to -15 000 cm H 2 O (wilting point). Two simple power-function k a (e) models, both with measured k, at -100 cm H 2 O as a reference point, gave similar and good predictions of k a (e) between -10 and -1000 cm H 2 O. For one location comprising finely textured and humic Andisols, both models largely underpredicted k,(e) in dry soil (

This study investigated the mechanisms inducing autonomic dysreflexia due to enhanced bladder-to-vascular reflexes in rats with spinal cord injury (SCI). SCI was produced by the transection of the Th4–5 spinal cord in female Sprague–Dawley rats. At 4 weeks after SCI, changes in blood pressure during graded increases in intravesical pressure (20–60 cm H2O) were measured in spinal-intact (SI) and SCI rats under urethane anesthesia. In five animals, effects of C-fiber desensitization induced by intravesical application of resiniferatoxin (RTX), a TRPV1 agonist, on the bladder-to-vascular reflex were also examined. Nerve growth factor (NGF) levels of mucosa and detrusor muscle layers of the bladder were measured by enzyme-linked immunosorbent assay. The expression levels of TRPV1 and TRPA1 channels were also examined in laser captured bladder afferent neurons obtained from L6 DRG, which were labeled by DiI injected into the bladder wall. In SI and SCI rats, systemic arterial blood pressure was increased in a pressure-dependent manner during increases in the intravesical pressure, with significantly higher blood pressure elevation at the intravesical pressure of 20 cm H2O in SCI rats vs SI rats. The arterial blood pressure responses to bladder distention were significantly reduced by RTX-induced desensitization of C-fiber bladder afferent pathways. SCI rats had higher NGF protein levels in the bladder and higher TRPV1 and TRPA1 mRNA levels in bladder afferent neurons compared with SI rats. The bladder-to-vascular reflex induced by TRPV1-expressing C-fiber afferents during bladder distention is enhanced after SCI in association with increased expression of NGF in the bladder and TRP channels in bladder afferent neurons.

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.

Copyright © 2014 Amaral, Brito, Chobanyan, Yoshikawa, Yokokura, Van Vactor and Gama-Carvalho. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order to achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of "biological quality" are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system (CNS) of a Drosophila transgenic strain with neuronal-specific RNAi of an ubiquitous gene was profiled using RNA-seq. After observing the existence of an unusual variance in our dataset, we showed that the expression profile of a small panel of marker genes, including GAL4 under control of a tissue specific driver, can identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We further analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we showed that trimming 5' ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes with a potential positive impact in the incurrence of systematic technical errors., Andreia J. Amaral was supported by a Marie Curie European Integration Grant (PERG-GA-2009-256595), an FCT post-doctoral grant (SFRH/BPD/65976/2009), and an EMBO long term fellowship (ALTF33-2010). Andreia J. Amaral, Francisco F. Brito, and Margarida Gama-Carvalho were further supported by FCT—PEst-OE/BIA/UI4046/2011 funds. Tamar Chobanyan, Seiko Yoshikawa, and Takakazu Yokokura were supported by OIST; David Van Vactor was also supported by grants from NINDS.

EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure–activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR.

Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro(274), which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases.

The soil gas diffusion coefficient ( D p) and its variations with soil air content (e) and soil water matric potential (ψ) control vadose zone transport and emissions of volatile organic chemicals and greenhouse gases. This study revisits the 1904 Buckingham power-law model where D p is proportional to e X , with X characterizing the tortuosity and connectivity of air-filled pore space. One hundred years later, most models linking D p(e) to soil water retention and pore size distribution still assume that the pore connectivity factor, X , is a constant for a given soil. We show that X varies strongly with both e and matric potential [given as pF = log(−ψ, cm H2O)] for individual soils ranging from undisturbed sand to aggregated volcanic ash soils (Andisols). For Andisols with bimodal pore size distribution, the X –pF function appears symmetrical. The minimum X value is typically around 2 and was observed close to ψ of −1000 cm H2O (pF 3) when interaggregate voids are drained. To link D p with bimodal pore size distribution, we coupled a two-region van Genuchten soil water retention model with the Buckingham D p(e) model, assuming X to vary symmetrically around a given pF. The coupled model well described D p as a function of both e and ψ for both repacked and undisturbed Andisols and for other soil types. By merely using average values of the three constants in the proposed symmetrical X –pF expression, predictions of D p were better than with traditional models.

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 A crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nico­tinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported. The monomeric structure comprises the two domains typical of NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The crystal structure and analytical ultracentrifugation results revealed dimer formation. In the NADP(H)-binding site, the pyrophosphate moiety and the 2′-­phosphoadenosine moiety are recognized by the glycine-rich loop (residues 157–162) and by loop residues 180–182, respectively. Furthermore, the present study revealed that P. horikoshii glyoxylate reductase contains aromatic clusters and has a larger number of ion pairs and a lower percentage of hydrophobic accessible surface area than its mesophilic homologues, suggesting its thermostability mechanism.

Air and water permeabilities and gas diffusivity as functions of soil fluid phase (air or water) contents are governing chemical transport and fate processes in the vadose zone, and have frequently been identified as the three main transport parameters determining time and efficiency during soil vapor extraction (soil venting) at polluted soil sites. A mathematically flexible function that can accurately describe data for both soil water retention and all three transport parameters as functions of fluid phase contents in undisturbed soil with bi- or multimodal pore structure is required for numerical simulation studies. In this study, a bimodal probability law (BPL) model with a total of six fitting parameters is compared with new and literature data for soil water retention and the three transport parameters measured on undisturbed soils. Saturated and unsaturated hydraulic conductivity (K) at six matric potentials (pF) were measured on four volcanic ash soils (Andisols) where data for soil water retention, air permeability (ka), and relative gas diffusivity (Dp/D0) were already available. The BPL model accurately described data for soil water retention and the three transport parameters (K, ka, Dp/D0) for the four Andisols and other soils with bimodal porosity behavior. BPL model parameters did not correlate well between transport processes, however, an existing model for relating saturated hydraulic conductivity to air permeability at 2100 cm H2O matric potential performed well for 10 bimodal Andisols. Results indicate that relations between fluid phase transport parameters at given matric potentials may be valid across soil types and useful in the BPL model.

The human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7) is involved in protein degradation, including a process known as endoplasmic reticulum-associated degradation (ERAD). The crystal structure of human UBE2G2/UBC7 was solved at 2.56 angstroms resolution. The UBE2G2 structure comprises a single domain consisting of an antiparallel beta-sheet with four strands, five alpha-helices and two 3(10)-helices. Structural comparison of human UBE2G2 with yeast Ubc7 indicated that the overall structures are similar except for the long loop region and the C-terminal helix. Superimposition of UBE2G2 on UbcH7 in a c-Cbl-UbcH7-ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way to UbcH7. In addition, the extra loop region of UBE2G2 may interact with the RING domain or its neighbouring region and may be involved in the binding specificity and stability.

Diffusion processes in the soil water and air phases often govern transport and fate of nutrients, pesticides, and toxic chemicals in the vadose zone. This final paper in a 10-part series on diffusion-reaction processes in soils concerns the development of a unifying model platform for predicting so