Computational Analysis of Activation of Dimerized Epidermal Growth Factor Receptor Kinase Using the String Method and Markov State Model.

Epidermal growth factor receptor (EGFR) activation is accompanied by dimerization. During the activation of the intracellular kinase domain, two EGFR kinases form an asymmetric dimer, and one side of the dimer (receiver) is activated. Using the string method and Markov state model (MSM), we performed a computational analysis of the structural changes in the activation of the EGFR dimer in this study. The string method reveals the minimum free-energy pathway (MFEP) from the inactive to active structure. The MSM was constructed from numerous trajectories of molecular dynamics simulations around the MFEP, which revealed the free-energy map of structural changes. In the activation of the receiver kinase, the unfolding of the activation loop (A-loop) is followed by the rearrangement of the C-helix, as observed in other kinases. However, unlike other kinases, the free-energy map of EGFR at the asymmetric dimer showed that the active state yielded the highest stability and revealed how interactions at the dimer interface induced receiver activation. As the H-helix of the activator approaches the C-helix of the receiver during activation, the A-loop unfolds. Subsequently, L782 of the receiver enters the pocket between the G- and H-helices of the activator, leading to a rearrangement of the hydrophobic residues around L782 of the receiver, which constitutes a structural rearrangement of the C-helix of the receiver from an outward to an inner position. The MSM analysis revealed long-time scale trajectories via kinetic Monte Carlo.

Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-HAD superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate-specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Construction of multiple metagenome assembled genomes containing carbon monoxide dehydrogenases from anaerobic carbon monoxide enrichment cultures.

Despite its toxicity to many organisms, including most prokaryotes, carbon monoxide (CO) is utilized by some aerobic and anaerobic prokaryotes. Hydrogenogenic CO utilizers employ carbon monoxide dehydrogenase (CODH) and energy-converting hydrogenase (ECH) to oxidize CO and reduce protons to produce H2. Those prokaryotes constitute a rare biosphere and are difficult to detect even with PCR amplification and with metagenomic analyses. In this study, anaerobic CO-enrichment cultures followed by construction of metagenome assembled genomes (MAGs) detected high-quality MAGs from potential hydrogenogenic CO utilizers. Of 32 MAGs constructed, 5 were potential CO utilizer harboring CODH genes. Of the five MAGs, two were classified into the genus Thermolithobacter on the basis of 16S rRNA sequence identity, related to Carboxydocella tharmautotrophica 41, with an average nucleotide identity (ANI) of approximately 72%. Additionally, two were related to Geoglobus acetivorans with ANI values ranging from 75 to 77% to G. acetivorans SBH6, and one MAG was identified as Desulfotomaculum kuznetsovii with an ANI > 96% to D. kuznetsovii DSM 6115. The two Thermolithobacter MAGs identified in this study contained CODH-ECH gene clusters, and were therefore identified as potential hydrogenogenic CO utilizers. However, these MAGs harbored three CODH gene clusters that showed distinct physiological functions in addition to CODH-ECH gene clusters. In total, the five potential CO utilizer MAGs contained sixteen CODH genes. Among those CODHs, four sets did not cluster with any known CODH protein sequences (with an identity of > 90%), and the CODH database was expanded.

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study.

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different.

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases.

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO2-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A-G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.

Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers.

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Anaerobic and hydrogenogenic carbon monoxide-oxidizing prokaryotes: Versatile microbial conversion of a toxic gas into an available energy.

Carbon monoxide (CO) is a gas that is toxic to various organisms including humans and even microbes; however, it has low redox potential, which can fuel certain microbes, namely, CO oxidizers. Hydrogenogenic CO oxidizers utilize an energy conservation system via a CO dehydrogenase/energy-converting hydrogenase complex to produce hydrogen gas, a zero emission fuel, by CO oxidation coupled with proton reduction. Biochemical and molecular biological studies using a few model organisms have revealed their enzymatic reactions and transcriptional response mechanisms using CO. Biotechnological studies for CO-dependent hydrogen production have also been carried out with these model organisms. In this chapter, we review recent advances in the studies of these microbes, which reveal their unique and versatile metabolic profiles and provides future perspectives on ecological roles and biotechnological applications. Over the past decade, the number of isolates has doubled (37 isolates in 5 phyla, 20 genera, and 32 species). Some of the recently isolated ones show broad specificity to electron acceptors. Moreover, accumulating genomic information predicts their unique physiologies and reveals their phylogenomic relationships with novel potential hydrogenogenic CO oxidizers. Combined with genomic database surveys, a molecular ecological study has unveiled the wide distribution and low abundance of these microbes. Finally, recent biotechnological applications of hydrogenogenic CO oxidizers have been achieved via diverse approaches (e.g., metabolic engineering and co-cultivation), and the identification of thermophilic facultative anaerobic CO oxidizers will promote industrial applications as oxygen-tolerant biocatalysts for efficient hydrogen production by genomic engineering.

Carbon monoxide-dependent transcriptional changes in a thermophilic, carbon monoxide-utilizing, hydrogen-evolving bacterium Calderihabitans maritimus KKC1 revealed by transcriptomic analysis.

Calderihabitans maritimus KKC1 is a thermophilic, carbon monoxide (CO)-utilizing, hydrogen-evolving bacterium that harbors seven cooS genes for anaerobic CO dehydrogenases and six hyd genes for [NiFe] hydrogenases and capable of using a variety of electron acceptors coupled to CO oxidation. To understand the relationships among these unique features and the transcriptional adaptation of the organism to CO, we performed a transcriptome analysis of C. maritimus KKC1 grown under 100% CO and N2 conditions. Of its 3114 genes, 58 and 32 genes were significantly upregulated and downregulated in the presence of CO, respectively. A cooS-ech gene cluster, an "orphan" cooS gene, and bidirectional hyd genes were upregulated under CO, whereas hydrogen-uptake hyd genes were downregulated. Transcriptional changes in anaerobic respiratory genes supported the broad usage of electron acceptors in C. maritimus KKC1 under CO metabolism. Overall, the majority of the differentially expressed genes were oxidoreductase-like genes, suggesting metabolic adaptation to the cellular redox change upon CO oxidation. Moreover, our results suggest a transcriptional response mechanism to CO that involves multiple transcription factors, as well as a CO-responsive transcriptional activator (CooA). Our findings shed light on the diverse mechanisms for transcriptional and metabolic adaptations to CO in CO-utilizing and hydrogen-evolving bacteria.

Hydration properties of a protein at low and high pressures: Physics of pressure denaturation.

Using experimentally determined structures of ubiquitin at 1 and 3000 bar, we generate sufficiently large ensembles of model structures in the native and pressure-induced (denatured) states by means of molecular dynamics simulations with explicit water. We calculate the values of a free-energy function (FEF), which comprises the hydration free energy (HFE) and the intramolecular (conformational) energy and entropy, for the two states at 1 and 3000 bar. The HFE and the conformational entropy, respectively, are calculated using our statistical-mechanical method, which has recently been shown to be accurate, and the Boltzmann-quasi-harmonic method. The HFE is decomposed into a variety of physically insightful components. We show that the FEF of the native state is lower than that of the denatured state at 1 bar, whereas the opposite is true at 3000 bar, thus being successful in reproducing the pressure denaturation. We argue that the following two quantities of hydration play essential roles in the denaturation: the WASA-dependent term in the water-entropy loss upon cavity creation for accommodating the protein (WASA is the water-accessible surface area of the cavity) and the protein-water Lennard-Jones interaction energy. At a high pressure, the mitigation of the serious water crowding in the system is the most important, and the WASA needs to be sufficiently enlarged with the increase in the excluded-volume being kept as small as possible. The denatured structure thus induced is characterized by the water penetration into the protein interior. The pressure denaturation is accompanied by a significantly large gain of water entropy.

An accurate and rapid method for calculating hydration free energies of a variety of solutes including proteins.

A new method is developed for calculating hydration free energies (HFEs) of polyatomic solutes. The solute insertion is decomposed into the creation of a cavity in water matching the geometric characteristics of the solute at the atomic level (process 1) and the incorporation of solute-water van der Waals and electrostatic interactions (process 2). The angle-dependent integral equation theory combined with our morphometric approach and the three-dimensional interaction site model theory are applied to processes 1 and 2, respectively. Neither a stage of training nor parameterization is necessitated. For solutes with various sizes including proteins, the HFEs calculated by the new method are compared to those obtained using a molecular dynamics simulation based on solution theory in energy representation (the ER method developed by Matubayasi and co-workers), currently the most reliable tool. The agreement is very good especially for proteins. The new method is characterized by the following: The calculation can rapidly be finished; a solute possessing a significantly large total charge can be handled without difficulty; and since it yields not only the HFE but also its many physically insightful energetic and entropic components, it is best suited to the elucidation of mechanisms of diverse phenomena such as the receptor-ligand binding, different types of molecular recognition, and protein folding, denaturation, and association.

Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus, we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA genes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants of T. thermophilus showed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor. T. thermophilus NurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants of nurA and herA in the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition, T. thermophilus NurA and HerA interacted with each other in vitro and in vivo, forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links in T. thermophilus cells. This cellular function is distinctly different from that of archaeal NurA and HerA.IMPORTANCE Many nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previous in vitro analyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performed in vivo analyses to address the cellular function of nurA and herA in an extremely thermophilic bacterium, Thermus thermophilus As a result, T. thermophilus NurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases.

The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair.

Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair.

Universal effects of solvent species on the stabilized structure of a protein.

We investigate the effects of solvent specificities on the stability of the native structure (NS) of a protein on the basis of our free-energy function (FEF). We use CPB-bromodomain (CBP-BD) and apoplastocyanin (apoPC) as representatives of the protein universe and water, methanol, ethanol, and cyclohexane as solvents. The NSs of CBP-BD and apoPC consist of 66% α-helices and of 35% ß-sheets and 4% α-helices, respectively. In order to assess the structural stability of a given protein immersed in each solvent, we contrast the FEF of its NS against that of a number of artificially created, misfolded decoys possessing the same amino-acid sequence but significantly different topology and α-helix and ß-sheet contents. In the FEF, we compute the solvation entropy using the morphometric approach combined with the integral equation theories, and the change in electrostatic (ES) energy upon the folding is obtained by an explicit atomistic but simplified calculation. The ES energy change is represented by the break of protein-solvent hydrogen bonds (HBs), formation of protein intramolecular HBs, and recovery of solvent-solvent HBs. Protein-solvent and solvent-solvent HBs are absent in cyclohexane. We are thus able to separately evaluate the contributions to the structural stability from the entropic and energetic components. We find that for both CBP-BD and apoPC, the energetic component dominates in methanol, ethanol, and cyclohexane, with the most stable structures in these solvents sharing the same characteristics described as an association of α-helices. In particular, those in the two alcohols are identical. In water, the entropic component is as strong as or even stronger than the energetic one, with a large gain of translational, configurational entropy of water becoming crucially important so that the relative contents of α-helix and ß-sheet and the content of total secondary structures are carefully selected to achieve sufficiently close packing of side chains. If the energetic component is excluded for a protein in water, the priority is given to closest side-chain packing, giving rise to the formation of a structure with very low α-helix and ß-sheet contents. Our analysis, which requires minimal computational effort, can be applied to any protein immersed in any solvent and provides robust predictions that are quite consistent with the experimental observations for proteins in different solvent environments, thus paving the way toward a more detailed understanding of the folding process.

Onboard Image Processing System for Hyperspectral Sensor.

Onboard image processing systems for a hyperspectral sensor have been developed in order to maximize image data transmission efficiency for large volume and high speed data downlink capacity. Since more than 100 channels are required for hyperspectral sensors on Earth observation satellites, fast and small-footprint lossless image compression capability is essential for reducing the size and weight of a sensor system. A fast lossless image compression algorithm has been developed, and is implemented in the onboard correction circuitry of sensitivity and linearity of Complementary Metal Oxide Semiconductor (CMOS) sensors in order to maximize the compression ratio. The employed image compression method is based on Fast, Efficient, Lossless Image compression System (FELICS), which is a hierarchical predictive coding method with resolution scaling. To improve FELICS's performance of image decorrelation and entropy coding, we apply a two-dimensional interpolation prediction and adaptive Golomb-Rice coding. It supports progressive decompression using resolution scaling while still maintaining superior performance measured as speed and complexity. Coding efficiency and compression speed enlarge the effective capacity of signal transmission channels, which lead to reducing onboard hardware by multiplexing sensor signals into a reduced number of compression circuits. The circuitry is embedded into the data formatter of the sensor system without adding size, weight, power consumption, and fabrication cost.

[Association Between the pH and Clinicopathological Characteristics of Lung Cancer Tissue].

Recent studies have shown that cancer cells surviving in a microenvironment characterized by hypoxia, low pH, and low glucose level have an ability to adapt to the adverse conditions. We measured the pH in the central tumor area of primary lung cancer, and evaluated its association with clinicopathological factors. There was a negative correlation between the tumor size and pH;with an increase in the tumor size, the pH decreased. Cancer cells grow at a markedly low pH compared with the physiological environment. There is a possibility that this low pH is a microenvironment that is appropriate rather than adverse for the growth and development of cancer cells.

Structural insights of post-translational modification sites in the proteome of Thermus thermophilus.

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701-2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of 'nearby' and 'peripheral' phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.

[Inpatient advocacy under the new system of involuntary hospitalization].

I considered both procedural and substantive requirements for patient advocacy in the context of involuntary hospitalization, and reviewed the revised Act on Mental Health and Welfare for the Mentally Disabled. I concluded that the revised act does not satisfy either procedural or substantive requirements for the advocacy of patients. The revised act stipulates that it be reviewed after three years. Along with carefully monitoring implementation of the new law, we must also create systems for reviewing the introduction of advocators, as well as enhancements to psychiatric review boards.

Draft genome of Parageobacillus thermoglucosidasius, a member of hydrogenogenic carbon monoxide utilizers, isolated from a freshwater lake sediment.

Parageobacillus thermoglucosidasius is a facultatively anaerobic thermophile and possesses carbon monoxide dehydrogenase and hydrogenase for carbon monoxide (CO) oxidation and hydrogen production, respectively. In this study, we report a draft genome of P. thermoglucosidasius isolated from a freshwater sediment, expanding our knowledge on the distribution of CO utilizers.