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Over hundreds of millions of years, organisms have evolved architected structures via precise control over hierarchically assembled components, including the integration of dissimilar materials. One such example is found in the radula system of chitons, intertidal mollusks that feed on algae growing on the rock. Their radula consists of multiple rows of ultrahard teeth, each integrated with a foldable belt-like substrate via a stiff, yet flexible stylus, which is essential for efficient rasping during the feeding process. Here, we investigate the nano and micro-scale components and architectures as well as regional mechanical properties of the stylus, and their subsequent role during the rasping of Cryptochiton stelleri. Three important factors were determined to contribute to the regio-specific stiffness of the stylus: the presence of mineral components, highly oriented chitinous fibers, and a chemically cross-linked protein matrix. All these factors are varied throughout the stylus. There is a high mineral content on the trailing edge close to the tooth and a cross-linked matrix on the leading edge, both with orientational specific oriented chitin fibers that provide force transduction to the tooth. Conversely, there is a significant lack of mineral or cross-linked matrix in the proximal end as well as a low degree of fiber orientation, resulting in a flexible region that can accommodate torsion and flexure during rasping. Understanding the graded composite structure of the stylus and applying this unique design to various engineering fields such as soft robotics, biotechnology, and the medical industry, can inspire the production of high-performance materials.

Over hundreds of millions of years, organisms have derived specific sets of traits in response to common selection pressures that serve as guideposts for optimal biological designs. A prime example is the evolution of toughened structures in disparate lineages within plants, invertebrates, and vertebrates. Extremely tough structures can function much like armor, battering rams, or reinforcements that enhance the ability of organisms to win competitions, find mates, acquire food, escape predation, and withstand high winds or turbulent flow. From an engineering perspective, biological solutions are intriguing because they must work in a multifunctional context. An organism rarely can be optimally designed for only one function or one environmental condition. Some of these natural systems have developed well-orchestrated strategies, exemplified in the biological tissues of numerous animal and plant species, to synthesize and construct materials from a limited selection of available starting materials. The resulting structures display multiscale architectures with incredible fidelity and often exhibit properties that are similar, and frequently superior, to mechanical properties exhibited by many engineered materials. These biological systems have accomplished this feat through the demonstrated ability to tune size, morphology, crystallinity, phase, and orientation of minerals under benign processing conditions (i.e., near-neutral pH, room temperature, etc.) by establishing controlled synthesis and hierarchical 3D assembly of nano- to microscaled building blocks. These systems utilize organic-inorganic interactions and carefully controlled microenvironments that enable kinetic control during the synthesis of inorganic structures. This controlled synthesis and assembly requires orchestration of mineral transport and nucleation. The underlying organic framework, often consisting of polysaccharides and polypeptides, in these composites is critical in the spatial and temporal regulation of these processes. In fact, the organic framework is used not only to provide transport networks for mineral precursors to nucleation sites but also to precisely guide the formation and phase development of minerals and significantly improve the mechanical performance of otherwise brittle materials.Over the past 15 years, we have focused on a few of these extreme performing organisms, (Wang

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

Although the number of HIV-2-infected individuals is quite low in Japan, at least three groups of HIV-2 (A, B and CRF01_AB) have been detected thus far. In particular, CRF01_AB HIV-2 cases have been found only in limited areas, Cote d'Ivoire and Japan. Here, we demonstrate that Geenius HIV 1/2 Confirmatory Assay (Geenius, Bio-Rad Laboratories) is able to detect HIV-2 samples, including groups A, B and CRF01_AB, isolated in Japan.A total of 57 plasma samples, including three panels (Ⅰ: HIV-2-positive samples [n=9], Ⅱ: HIV-1 infection with HIV-2 antibody cross-reactivity samples [n=37], and Ⅲ: HIV negative with biological false-positive HIV-2 samples [n=11]) were tested by Geenius.Geenius determined Panel I to be "HIV-2 positive with/without HIV-1 cross-reactivity (n=4, respectively)", including HIV-2 group A and CRF01_AB. In the case with HIV-2 group B, all bands were detected, resulting in a Geenius interpretation of "HIV positive untypable". Geenius classified Panels II and III as "HIV-1 positive (n=37)" or "HIV negative (n=9)", "HIV indeterminate (n=1)" and "HIV-2 indeterminate (n=1)", suggesting 95.8% HIV-2 differentiation by Geenius.With Geenius, there were fewer false-positives for HIV-1/-2 negativity and fewer cross-reactions with HIV-2 among HIV-1-positive samples. Additionally, the assay could detect HIV-2 genetic group CRF01_AB. Geenius can be expected to be a useful diagnostic tool that is an alternative to conventional Western blotting.

In HIV-1-infected patients, antiretroviral therapy (ART) is a key factor that may impact commensal microbiota and cause the emergence of side effects. However, it is not fully understood how long-term ART regimens have diverse impacts on the microbial compositions over time. Here, we performed 16S ribosomal RNA gene sequencing of the fecal and salivary microbiomes in patients under different long-term ART. We found that ART, especially conventional nucleotide/nucleoside reverse transcriptase inhibitor (NRTI)-based ART, has remarkable impacts on fecal microbial diversity: decreased α-diversity and increased ß-diversity over time. In contrast, dynamic diversity changes in the salivary microbiome were not observed. Comparative analysis of bacterial genus compositions showed a propensity for Prevotella-enriched and Bacteroides-poor gut microbiotas in patients with ART over time. In addition, we observed a gradual reduction in Bacteroides but drastic increases in Succinivibrio and/or Megasphaera under conventional ART. These results suggest that ART, especially NRTI-based ART, has more suppressive impacts on microbiota composition and diversity in the gut than in the mouth, which potentially causes intestinal dysbiosis in patients. Therefore, NRTI-sparing ART, especially integrase strand transfer inhibitor (INSTI)- and/or non-nucleotide reverse transcriptase inhibitor (NNRTI)-containing regimens, might alleviate the burden of intestinal dysbiosis in HIV-1-infected patients under long-term ART.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory–based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

dl-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3 mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

Chitons are herbivorous invertebrates that use rows of ultrahard magnetite-based teeth connected to a flexible belt (radula) to rasp away algal deposits growing on and within rocky outcrops along coastlines around the world. Each tooth is attached to the radula by an organic structure (stylus) that provides mechanical support during feeding. However, the underlying structures within the stylus, and their subsequent function within the chiton have yet to be investigated. Here, we investigate the macrostructural architecture, the regional material and elemental distribution and subsequent nano-mechanical properties of the stylus from the Northern Pacific dwelling Cryptochiton stelleri. Using a combination of μ-CT imaging, optical and electron microscopy, as well as elemental analysis, we reveal that the stylus is a highly contoured tube, mainly composed of alpha-chitin fibers, with a complex density distribution. Nanoindentation reveals regiospecific and graded mechanical properties that can be correlated with both the elemental composition and material distribution. Finite element modeling shows that the unique macroscale architecture, material distribution and elemental gradients have been optimized to preserve the structural stability of this flexible, yet robust functionally-graded fiber-reinforced composite tube, providing effective function during rasping. Understanding these complex fiber-based structures offers promising blueprints for lightweight, multifunctional and integrated materials.

In June 2015, Diagnostic Reference Levels was drawn up in Japan. Therefore, the exposure control of the examinees is more important than ever. In this research, in order to grasp the AGD for each examinee, a basic examination of a method to measure X - ray output without changing the quality of the beam was made. In order not to change the quality of the radiation, scattered radiation from the added filter is used and the added filter is mounted in the X-ray source device, so it is necessary to dispose the dosimeter also in the X-ray source device. It was measured with a semiconductor dosimeter. The measurement conditions, that are likely optimal judging from the examination results, are as follows: (1) The semiconductor dosimeter is to be cooled constantly due to its temperature characteristics. (2) The straight line connecting the added filter center and the semiconductor dosimeter?s detector center is to be orthogonal to the detection plane of the dosimeter?s detector constantly. (3) The dosimeter?s detector is to be opposed, at an angle not blocking the primary X-ray, to the added filter as much as possible. It is likely possible to calculate the X-ray output in mammographic X-ray apparatuses by multiplying the dose of the scattered radiation measured in the layout above by the respective calibration constant of each target/added filter combination. In addition, we believe that it is possible to grasp AGD by examinee by multiplying the X-ray output by coefficients additionally.

The denticle caps of chiton teeth exhibit the largest hardness and stiffness among any biological minerals in the world. They consist of a composite structure of highly oriented crystalline nanorods of magnetite surrounded by a veneer of organic. The ultrahard teeth of the chiton are the first reported example of biologically formed magnetite, and its formation mechanisms have been of great interest to researchers for decades. Recently, using modern nanomechanical characterization techniques, it was shown that chiton teeth exhibit the largest hardness and stiffness of any biological mineral. The predicted abrasion resistance of chiton teeth against a blunt contact was reported to be even higher than that of zirconia. Therefore, the elucidation of chiton teeth synthetic processes could help us learn how to develop novel abrasion-resistant materials and environmentally benign processes for the production of iron oxides and other nanostructured materials. In order to understand the chiton teeth formation, proteomic analyses of tooth proteins as well as detailed structural analyses of mature and developing teeth were conducted. Based on the results obtained from these analyses, we discuss the underlying mechanisms of iron oxide biomineralization in chiton teeth.

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-14C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The KM and kcat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min−1, respectively.

Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4+ T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings’ genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient’s T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

Summary Fistulifera sp. strain JPCC DA0580, a marine pennate diatom, contains extremely high levels of intracellular triglyceride and has been suggested as a promising source of feedstock for biodiesel fuels. JPCC DA0580 was isolated from a mangrove swamp located in Sumiyo Bay below the mouths of the Sumiyo and Yakugachi Rivers in Amami-Ohshima, Kagoshima, Japan. In this study, the morphology and the 18S rDNA sequence of JPCC DA0580 were compared with those of other Fistulifera strains. JPCC DA0580 possesses morphological characters of the genus Fistulifera, namely lightly silicified frustules, a distinct median costa (raphe sternum), and a wart-like central pore (fistula). Morphometric analysis revealed that JPCC DA0580 differs from other Fistulifera species by the presence of a valve with coarser striation and coarser areolation. On the basis of 18S rDNA phylogeny, JPCC DA0580 formed a well-supported clade with other members of the Fistulifera species complex, although the number of nucleotide substitutions was highest in JPCC DA0580. Our results led us to propose the taxonomic name Fistulifera solaris sp. nov. for JPCC DA0580.

Among the proteins localized on the cell wall (frustule) of diatoms (frustule-associated proteins), several proteins tightly associated with the cell wall have been implicated in frustule formation. These proteins include diatom-specific unique serine- and lysine-rich sequences represented by silaffins. Taking advantage of available genome information, we used a recently described bioinformatics approach to screen silaffin-like proteins rich in serine and lysine from the genome of the marine pennate diatom Fistulifera sp. strain JPCC DA0580 and identified 7 proteins. All of the proteins shared a sequence motif called the XGXG domain, which was also confirmed in a silaffin-like protein identified in other diatoms. In vivo localization analysis revealed that one of the identified proteins, G7408, occurs throughout the frustule with a slightly uneven distribution. This novel frustule-associated protein could be a useful tool to elucidate the mechanism of biosilica formation in diatoms and to functionalize this strain for future biotechnological applications.

A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60 % w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6 μm in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production.

During mineralization, the hard outer magnetite-containing shell of the radular teeth of Cryptochiton stelleri undergoes four distinct stages of structural and phase transformations: (i) the formation of a crystalline α -chitin organic matrix that forms the structural framework of the non-mineralized teeth, (ii) the templated synthesis of ferrihydrite crystal aggregates along these organic fi bers, (iii) subsequent solid state phase transformation from ferrihydrite to magnetite, and (iv) progressive magnetite crystal growth to form continuous parallel rods within the mature teeth. The underlying α -chitin organic matrix appears to infl uence magnetite crystal aggregate density and the diameter and curvature of the resulting rods, both of which likely play critical roles in determining the local mechanical properties of the mature radular teeth.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

The biomineralized radular teeth of chitons are known to consist of iron-based magnetic crystals, associated with the maximum hardness and stiffness of any biomineral. Based on our transmission electron microscopy analysis of partially mineralized teeth, we suggest that the organic matrix within the teeth controls the iron oxide nucleation. Thus, we used Nano-LC-MS to perform a proteomic analysis of the organic matrix in radular teeth of the chiton Cryptochiton stelleri in order to identify the proteins involved in the biomineralization process. Since the genome sequence of C. stelleri is not available, cross-species similarity searching and de novo peptide sequencing were used to screen the proteins. Our results indicate that several proteins were dominant in the mineralized part of the radular teeth, amongst which, myoglobin and a highly acidic peptide were identified as possibly involved in the biomineralization process.

Many textiles have been often used in marine environments for a wide variety of purposes. Consequently, effective and clean methods to prevent biofouling in these textiles are needed. In this study, a nylon mesh doped with AgI was examined for its antifouling performance. The nylon mesh was iodinated with an iodine–potassium iodide aqueous solution and subsequently immersed in silver nitrate solution to form AgI particles in the fiber. SEM-EDX analysis indicated that the AgI crystals were highly incorporated into the nylon 6,6 polymer (173 wt% gain). The bactericidal activity of AgI-doped nylon mesh was more than 200 times higher than that of silver-coated nylon mesh. Furthermore, continuous prevention of biofouling in the coastal marine environment was attained for 437 days by using the AgI-doped nylon mesh. Since the concentration of the released silver ions from the nylon mesh was estimated to be less than 1 ppb, the proposed nylon mesh is useful as an effective and clean method for prevention of biofouling in polymer textiles.

We propose a copper iodide (CuI)-doped nylon mesh prepared using polyiodide ions as a precursor toward anti-biofouling polymer textile. The CuI-doped nylon mesh was subjected to the prevention of biofouling in marine environments. The attachment of the marine organisms was markedly inhibited on the CuI-doped nylon mesh surface until 249 days. Scanning electron microscopy-energy dispersive X-ray analysis indicated that copper compounds were maintained in the nylon mesh after the field experiment, although copper content in the nylon mesh was reduced. Therefore, the copper ions slowly dissolved from nylon mesh will contribute to the long-term prevention of biofouling. Furthermore, electron spin resonance analysis revealed the generation of reactive oxygen species (ROS) from CuI-doped nylon mesh after the field experiment. One of the possibilities for toxic action of copper ions will be the direct effect of Cu+ -induced ROS on biofilm forming on nylon mesh surface. The proposed polymer textile can be applied to fishing and aquafarming nets, mooring rope for ship, or silt fence to restrict polluted water in marine environments.

Recent molecular studies on magnetotactic bacteria have identified a number of proteins associated with bacterial magnetites (magnetosomes) and elucidated their importance in magnetite biomineralisation. However, these analyses were limited to magnetotactic bacterial strains belonging to the alpha-subclass of Proteobacteria. We performed a proteomic analysis of magnetosome membrane proteins in Desulfovibrio magneticus strain RS-1, which is phylogenetically classified as a member of the delta-Proteobacteria. In the analysis, the identified proteins were classified based on their putative functions and compared with the proteins from the other magnetotactic bacteria, Magnetospirillum magneticum AMB-1 and M. gryphiswaldense MSR-1. Three magnetosome-specific proteins, MamA (Mms24), MamK, and MamM, were identified in strains RS-1, AMB-1, and MSR-1. Furthermore, genes encoding ten magnetosome membrane proteins, including novel proteins, were assigned to a putative magnetosome island that contains subsets of genes essential for magnetosome formation. The collagen-like protein and putative iron-binding proteins, which are considered to play key roles in magnetite crystal formation, were identified as specific proteins in strain RS-1. Furthermore, genes encoding two homologous proteins of Magnetococcus MC-1 were assigned to a cryptic plasmid of strain RS-1. The newly identified magnetosome membrane proteins might contribute to the formation of the unique irregular, bullet-shaped crystals in this microorganism.

The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCE HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni–Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD .

Supplemental Data; International audience; Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Mammalian homologues, Sephs1 and Sephs2, differ greatly in their ability to complement selD mutation in E. coli. Sephs2 is a selenoenyzme that has the Sec60 residue in the active site. Sephs2 retained the in vivo complementation when the Sec residue was replaced with Cys. Therefore, Sephs2Cys gene allowed the production of the bacterial selenoenzyme, formate dehydrogenases, in a selD mutant of E. coli. In contrast, Sephs1 has a Thr residue at the corresponding active site, and is an inactive catalysis producing no complementation effect in E. coli selD mutant. Although Sephs2 and Sephs1 share a high homology in their amino acid sequences, Sephs1-Thr29Cys mutant did not complement selD mutation in E. coli, suggesting that the lack of the active site Cys was not the sole reason for the inert catalysis. Sephs2 gene encodes an internal Pro-rich sequence spanning Glu43-Arg83 (designated as int2), while Sephs1 has a corresponding, but rather a shorter sequence Ala74-Ala114 (designated as int1). The in vivo complementation activity of Sephs2 was completely lost when the int2 sequence was substituted for the int1 sequence. Conversely, Sephs1 gained the SEPHS activity when the int1 region was replaced with int2 sequence in addition to the Thr to Cys substitution at the active site. Our results demonstrated for the first time that structural requirement of mammalian Sephs includes the proline-rich int2 region, although bacterial SEPHS including E. coli SelD has not such an internal sequence region corresponding to the int2 sequence. Interestingly, co-expression of Sephs1 gene under varied concentrations of L-arabinose lead to gradual suppression of selD complementation by Sephs2Cys gene in E. coli. The hypothesis that the inactive homologue Sephs1 can suppress Sephs2Cys activity is also supported by in vitro pull-down assay and yeast-2-hybrid assay, which both indicated binding of Sephs1 to Seph2Cys.

For biodiesel production from microalgae, it is desirable to understand the entire triacylglycerol (TAG) metabolism. TAG accumulation occurs in oil bodies, and although oil body-associated proteins could play important roles in TAG metabolism, only a few microalgal species have been studied by a comprehensive analysis. Diatoms are microalgae that are promising producers of biodiesel, on which such proteomics analysis has not been conducted to date. Herein, we identified oil body-associated proteins in the oleaginous diatom Fistulifera sp. strain JPCC DA0580. The oil body fraction was separated by cell disruption with beads beating and subsequent ultracentrifugation. Contaminating factors could be removed by comparing proteins from the oil body and the soluble fractions. This novel strategy successfully revealed 15 proteins as oil body-associated protein candidates. Among them, two proteins, which were parts of proteins predicted to have transmembrane domains, were indeed confirmed to specifically localize to the oil bodies in this strain by observation of GFP fusion proteins. One (predicted to be a potassium channel) was also detected from the ER, suggesting that oil bodies might originate from the ER. By utilizing this novel subtraction method, we succeeded in identifying the oil body-associated proteins in the diatom for the first time.

The present study involved the designing of a culture process and the evaluation of productivity of oil products from a highly oleaginous marine diatom, Fistulifera sp. JPCC DA0580, which had been cultured in a commercial-scale factory. The culture facility had a capacity of 48,000 L and held 96 flat-type 500-L photobioreactors (PBRs) equipped with artificial light, which secures a stable, perennial supply of the products. A 10 days culture that had reached a cell density of 6.5 g dry weight L(-1) possessing a cellular oil content of 48% (wt/wt) was found to provide the highest oil yield. On considering a production area of 1500 m(2), annual algal mass and oil productivity is 68.7 and 33.3 t ha(-1) year(-1), respectively. This study thus provides a reproducible prediction of a theoretical maximum oil yield from a highly oleaginous microalgal strain based on industrially practical production area.

This study demonstrated the antiviral properties of copper iodide (CuI) nanoparticles against the non-enveloped virus feline calicivirus (FCV) as a surrogate for human norovirus. The effect of CuI nanoparticles on FCV infectivity to Crandell-Rees feline kidney (CRFK) cells was elucidated. The infectivity of FCV to CRFK cells was greatly reduced by 7 orders of magnitude at 1000μgml(-1) CuI nanoparticles. At the conditions, electron spin resonance (ESR) analysis proved hydroxyl radical production in CuI nanoparticle suspension. Furthermore, amino acid oxidation in the viral capsid protein of FCV was determined by nanoflow liquid chromatography-mass spectrometric (nano LC-MS) analysis. The use of CuI nanoparticles showed extremely high antiviral activity against FCV. The high antiviral property of CuI nanoparticles was attributed to Cu(+), followed by ROS generation and subsequent capsid protein oxidation. CuI nanoparticles could be proposed as useful sources of a continuous supply of Cu(+) ions for efficient virus inactivation. Furthermore, this study brings new insights into toxic actions of copper iodide nanoparticles against viruses.

Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein–magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.