Immunogenicity and safety of adsorbed diphtheria-purified pertussis-tetanus-inactivated polio (Sabin strain)-Haemophilus type b conjugate combined vaccine (DPT-IPV-Hib) in healthy Japanese Infants ≥ 2 and < 43 months of Age: A phase III, multicenter, active controlled, assessor-blinded, randomized, parallel-group study.

OBJECTIVE: This study investigated the immunogenicity and safety of a pentavalent vaccine Gobik (DPT-IPV-Haemophilus influenzae type b [Hib]) in healthy Japanese infants aged ≥ 2 and < 43 months using a concomitant vaccination with ActHIB® (Hib) and Tetrabik (DPT-IPV) as a comparator. METHODS: This study was conducted as a phase 3, multicenter, active controlled, assessor-blinded, randomized, parallel-group study. Participants received a total of 4 subcutaneous doses (3 primary immunization doses and a booster dose) of either the experimental drug (DPT-IPV-Hib) or the active comparator (Hib + DPT-IPV). The primary endpoints were the anti-PRP antibody prevalence rate with ≥ 1 µg/mL, and the antibody prevalence rates against pertussis, diphtheria toxin, tetanus toxin, and attenuated poliovirus after the primary immunization. RESULTS: In 267 randomized participants (133 in the DPT-IPV-Hib group and 134 in the Hib + DPT-IPV group), the antibody prevalence rates after the primary immunization in both groups were 100.0 % and 88.7 % for anti-PRP antibody with ≥ 1 µg/mL, 99.2 % and 98.5 % against diphtheria toxin, and 100.0 % and 99.2 % against tetanus toxin, respectively. The antibody prevalence rates against pertussis and attenuated poliovirus were 100.0 % in both groups. The non-inferiority of the DPT-IPV-Hib group to the Hib + DPT-IPV group was verified for all measured antibodies. In both groups, all the GMTs of antibodies after the primary immunization were higher than those before the first dose, and those after the booster dose were higher than those after the primary immunization. No safety issues were identified. CONCLUSION: A single-agent Gobik, the first DPT-IPV-Hib pentavalent vaccine approved in Japan, was confirmed to simultaneously provide primary and booster immunizations against Hib infection, pertussis, diphtheria, tetanus, and poliomyelitis and to have a preventive effect and safety comparable to concomitant vaccination with Hib (ActHIB®) and DPT-IPV quadrivalent vaccine (Tetrabik).

Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

A blue-shifted anion channelrhodopsin from the Colpodellida alga Vitrella brassicaformis.

Microbial rhodopsins, a family of photoreceptive membrane proteins containing the chromophore retinal, show a variety of light-dependent molecular functions. Channelrhodopsins work as light-gated ion channels and are widely utilized for optogenetics, which is a method for controlling neural activities by light. Since two cation channelrhodopsins were identified from the chlorophyte alga Chlamydomonas reinhardtii, recent advances in genomic research have revealed a wide variety of channelrhodopsins including anion channelrhodopsins (ACRs), describing their highly diversified molecular properties (e.g., spectral sensitivity, kinetics and ion selectivity). Here, we report two channelrhodopsin-like rhodopsins from the Colpodellida alga Vitrella brassicaformis, which are phylogenetically distinct from the known channelrhodopsins. Spectroscopic and electrophysiological analyses indicated that these rhodopsins are green- and blue-sensitive pigments (λmax = ~ 550 and ~ 440 nm) that exhibit light-dependent ion channeling activities. Detailed electrophysiological analysis revealed that one of them works as a monovalent anion (Cl-, Br- and NO3-) channel and we named it V. brassicaformis anion channelrhodopsin-2, VbACR2. Importantly, the absorption maximum of VbACR2 (~ 440 nm) is blue-shifted among the known ACRs. Thus, we identified the new blue-shifted ACR, which leads to the expansion of the molecular diversity of ACRs.

Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift.

The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named 'schizorhodopsin (SzR)' has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral 'blue-shift' due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage.

Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H+ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Draft Genome Sequence of Saccharospirillum sp. Strain MSK14-1, Isolated from Surface Seawater Collected at Aburatsubo Inlet in Japan.

Here, we report the draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan. The genome sequence of strain MSK14-1 should contribute to our understanding of the characteristics of the genus Saccharospirillum.

The complete mitochondrial genome of the longfin dragonfish Tactostoma macropus (Stomiiformes: Stomiidae).

The complete mitochondrial genome (mitogenome) was determined for the longfin dragonfish Tactostoma macropus, which is the first for the genus and the third within the family Stomiidae. The mitogenome sequence is 17,690 bp in length containing 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region, as in most fishes. The gene order of T. macropus showed an unreported deviation from the typical vertebrate one. Phylogenetic reconstruction using the maximum likelihood method placed T. macropus in the monophyletic Stomiiformes. Three stomiid species were recovered as a moderately supported clade in the phylogenetic tree.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis.

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco's modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.

Six month outcomes of an innovative weekly intergenerational day program with older adults and school-aged children in a Japanese urban community.

AIM: To describe the nature of the progression of intergenerational interactions among and between older people and children in a weekly intergenerational day program (IDP) in an urban community and to evaluate the older people's health-related quality of life (HRQOL) and depressive symptoms, compared to the program volunteers, as well as the children's perspectives of older people, during the first 6 months of the program's implementation. METHODS: This longitudinal study, with a convenience sample of older people (n = 14), program volunteers (n = 8), and school-aged children (n = 7), used mixed methods to analyze the results. Participant observations and interviews were used to describe the interactions between the generations over the 6 months. An ANOVA with repeated measures was used to determine the statistical effects over time (initially and at 3 months and 6 months) for HRQOL (Medical Outcomes Study 8-Item Short-Form Health Survey) and depression (Geriatric Depression Scale, GDS-15). Semantic differential scales identified the children's perspectives of older people. RESULTS: The intergenerational interactions were grouped into thirteen categories; for example, "The IDP provided a meaningful sense of place." The quality of life in relation to the mental health of the older people's group improved significantly between the first involvement and after 6 months, while the GDS-15 scores significantly decreased at the three time points in the more depressed older people's subgroup. The children's initial generally positive perspectives of older people showed no statistically significant change over time. CONCLUSION: The intergenerational interactions in the IDP yielded a meaningful place for both generations, improved the HRQOL of the older people's group, and decreased the depressive symptoms in the more depressed older people's subgroup.

Effect of wood properties on within-tree variation in ultrasonic wave velocity in softwood.

The radial variations in the velocity of longitudinal waves propagating through Japanese cedar and Japanese cypress were experimentally investigated. In addition, the tracheid length (TL), microfibril angle (MFA), air-dried density (AD), and moisture content (MC) were measured in order to determine the effect of wood properties on velocity variations within the wood trunk. For both species, the longitudinal wave velocities measured in the longitudinal direction (V(L)) exhibited minimum values near the pith. For Japanese cedar, V(L) increased from 3600m/s toward the bark and soon attained a constant value (=4500m/s). On the other hand, for Japanese cypress, V(L) kept increasing from 4000m/s near the pith to 4800m/s at the bark. These radial variations in V(L) coincided with those in the tracheid length. V(L) exhibited strong correlations with TL and MFA with a significant level of (p<0.01). These findings suggest that the TL and MFA greatly affect the radial variation in the ultrasonic wave velocity in softwood.

Epithelialization in oral mucous wound healing in terms of energy metabolism.

PURPOSE: The purpose of this study was to clarify the "healing capacity" of wounds of the oral mucosa in comparison to those of the skin, and to evaluate the wound healing mechanism of oral mucosa using a cytobiological approach from the aspect of energy metabolism in oral keratinocytes. MATERIALS AND METHODS: Samples of epidermal and oral keratinocytes collected at surgery and of cultured oral keratinocytes were used to analyze (1) by gas chromatography the composition of fatty acids (16:0, 18:2, 20:4) in the cell membranes of keratinocytes, (2) by immunohistochemical staining of GLUT-1 antibody and specific PAS staining the localization of glucose metabolism, and (3) by RT-PCR and Western blotting the expression of GLUT-1 mRNA and of protein in the keratinocytes of the basal and parabasal layers of each epithelial tissue. RESULTS: 1. The % composition of palmitic acid (16:0) was significantly higher in buccal mucosal keratinocytes (27.18+/-3.74%) and in the gingiva (23.00+/-1.40%) than in the epidermis (17.54+/-0.37%). 2. Immunohistochemical staining showed GLUT-1 protein in the skin to be expressed only in the bulge region of hair follicles and in the epidermal basal layer, and observed nearly throughout all epithelial cell layers in the oral mucosa. 3. PAS-positive cells were observed among differentiation-enhanced cells in the upper prickle layer in the oral mucosa. 4. The same results were obtained from RT-PCR and a Western blotting analysis. CONCLUSION: The present study demonstrated definite cytobiological evidence that the oral mucosa surpasses the skin in regard to its wound healing capacity.

Lipid analysis of normal dermis and hypertrophic scars.

Hypertrophic scars (HS) are a consequence of abnormal wound healing. We examined fatty acids that are contained within, and participate in, every reaction through the membrane; then, we analyzed the percentage composition of the fatty acids in deepithelialized normal dermis (ND) and HS. In vivo HS samples were obtained from six patients undergoing surgical excision, and ND samples from five patients undergoing skin grafting surgery for excess. In vitro, cultured fibroblasts from HS and ND were also analyzed. The percentage composition of fatty acids extracted from all the samples was analyzed. In vivo, arachidonic acid (20:4) was significantly more abundant in HS than in ND, in the phospholipids from both whole tissue and cell membranes. In vitro, there were no significant differences among ND, HS, and 10% fetal calf serum. The results suggest that HS formation does not necessarily involve simple excess of 20:4; however, there are considerable differences in the percentage composition of 20:4 between ND and HS. Arachidonic acid probably participates in the formation and maintenance of HS, whereas in vitro cultured fibroblasts are affected largely by fetal calf serum.

Effect of anisotropy on acoustoelastic birefringence in wood.

The ultrasonic velocity of shear waves propagating through radial direction of a wood plate specimen, transversely to the loading direction, was measured. By rotating an ultrasonic sensor, the oscillation direction of the shear waves was varied with respect to the wood plate axis and loading direction. The relationship between shear wave velocity and oscillation direction was examined to discuss the effect of anisotropy on the acoustoelastic birefringence in wood. The results obtained were summarized as follows. When the oscillation direction of the shear wave corresponded to the tangential direction of the wood specimen regardless of the stress direction, shear wave velocity decreased markedly and the relationship between shear wave velocity and rotation angle tended to become discontinuous. That is, when the shear waves oscillated in the anisotropic axis of the wood, the shear wave velocity peaked unlike in the case of oscillation in the stress direction. In an isotropic material (acrylic, aluminum 5052), on the contrary, when the shear waves oscillated in the stress direction of the specimen, the shear wave velocity peaked regardless of the main-axis direction of the specimen. On the basis of the discussion of these results, the ultrasonic shear wave propagating in wood under stress is confirmed to be polarized in the anisotropic axis of the wood.

Proapoptotic effect of a dietary supplement: water soluble chitosan activates caspase-8 and modulating death receptor expression.

The effect of water-soluble chitosan, a natural polymer used as a dietary supplement, on human bladder-tumor cells was investigated. Apoptotic morphological change was demonstrated by nuclear staining. Chitosan-treated cells showed elevation of caspase-8-like activity, but no significant elevation of caspase-9-like activity, which suggest that proapoptotic effect of chitosan is attributable to death receptor activation and not to activation of the mitochondria-cytochrome c pathway. Chitosan increased expression of TNF-R1, but decreased Fas expression. Use of monoclonal antibodies to inhibit death-receptor signal transduction did not attenuate the proapoptotic activity of chitosan. Examination of death-ligands revealed that TNFalpha mRNA expression was markedly increased by chitosan treatment while FasL mRNA was not affected. Although the direct interaction of chitosan with death receptors remains unidentified, the results suggest that its proapoptotic effect might be related to interaction with TNFalpha or TNF-R1.

Quantitative analysis of expression of NeuroD, GAP43 and receptor tyrosine kinase B in developing mouse olfactory neuroepithelium.

OBJECTIVE: The mammalian olfactory neuroepithelium (OE), which harbors olfactory receptor neurons (ORNs), has the unusual characteristic of continuous neurogenesis throughout its lifetime. This unique feature provides an excellent model for neuronal differentiation. Recently, we found dual-phase expression of NeuroD, a member of the basic helix-loop-helix transcription factor family, in the developing mouse OE, suggesting multiple roles of NeuroD during the development of mammalian ORNs. MATERIAL AND METHODS: In order to better understand the molecular mechanism of the development of ORNs, we performed quantitative analysis of expression of NeuroD, GAP43 and receptor tyrosine kinase B (TrkB), as well as 5-bromo-2'-deoxyuridine (BrdU)-labeled cells, in the developing mouse OE from gestational Day 10 to postnatal Day 28. RESULTS: During the embryonic period, NeuroD expression is mostly confined to the basal compartment. During the neonatal period, NeuroD expression is detected in two compartments: the middle compartment and the basal compartment. GAP43-expressing cells were located between these two NeuroD-positive layers. TrkB-expressing cells were located above the NeuroD-positive layer in the middle compartment. As the mice grew, the numbers of NeuroD-expressing cells and BrdU-labeled cells in the basal compartment significantly decreased, while the number of NeuroD-expressing cells in the middle compartment gradually increased. The number of TrkB-expressing cells dramatically increased. The number of GAP43-expressing cells also gradually increased. However, the relative proportion of GAP43 cells decreased as the OE developed. CONCLUSION: NeuroD is a useful molecular marker for studying olfactory neurogenesis.

Kainic acid-induced convulsions cause prolonged changes in the chondroitin sulfate proteoglycans neurocan and phosphacan in the limbic structures.

Systemic administration of kainic acid induces repeated convulsive seizures (KA convulsions) that result in neuropathological changes similar to temporal lobe epilepsy and the appearance of spontaneous recurrent seizures (SRS). The appearance of SRS is considered a result of the remodeling of neuronal networks following neuronal degeneration. We investigated the changes in chondroitin sulfate proteoglycans (CSPGs) in the limbic structures after KA convulsions in the rat using monoclonal antibodies 1G2, which recognizes full-length neurocan and the C-terminal half of neurocan, neurocan C, and 6B4, which recognize phosphacan and protein tyrosine phosphatase zeta. After KA convulsions, full-length neurocan appeared by 24 h and reached a peak by 48 to 72 h, whereas phosphacan decreased within 24 h in the hippocampus. In immunohistochemistry, neurocan increased in the limbic structures coincident with the appearance of reactive astrocytes. Phosphacan decreased coincident with pyramidal cell loss in the hippocampus, and the number of phosphacan-positive perineuronal nets around parvalbumin neurons decreased, whereas parvalbumin neurons were relatively conserved. In contrast, phosphacan increased in the entorhinal and piriform cortices in correlation with the severity of neuronal loss. Both neurocan and phosphacan recovered to the control level by 8 weeks after KA convulsions in some rats, but the changes in neurocan and phosphacan described above still persisted in more than half the rats. The results indicate that KA convulsions induce prolonged changes in neurocan and phosphacan similar to those in the developing rat brain and suggest a role of these CSPGs in the remodeling of neuronal networks related to the establishment or enhancement of epileptogenesis.