Involvement of Janus kinase-dependent Bcl-xL overexpression in steroid resistance of group 2 innate lymphoid cells in asthma.

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 µg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s.

Biochemical Aspects of Putative Aminotransferase Responsible for Converting Vanillin to Vanillylamine in the Capsaicinoid Biosynthesis Pathway in Capsicum Plants.

The biosynthesis pathway of capsaicinoids includes the conversion of vanillin to vanillylamine, where putative aminotransferase (pAMT) is thought to be the enzyme responsible in Capsicum plants. The objectives of this study were to prove that pAMT is the enzyme responsible for this conversion in plants and to clarify its catalytic properties using biochemical methods. Both an extract of habanero placenta and recombinant pAMT (rpAMT) constructed by using an Escherichia coli expression system were able to convert vanillin to vanillylamine in the presence of γ-aminobutyric acid as an amino donor and pyridoxal phosphate as a coenzyme. Conversion from vanillin to vanillylamine by the placenta extract was significantly attenuated by adding an anti-pAMT antibody to the reaction system. The amino donor specificity and affinity for vanillin of rpAMT were similar to those of the placenta extract. We thus confirmed that pAMT is the enzyme responsible for the conversion of vanillin to vanillylamine in capsaicinoid synthesis in Capsicum fruits. Therefore, we propose that pAMT should henceforth be named vanillin aminotransferase (VAMT).

An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

Expression of alcohol acyltransferase is a potential determinant of fruit volatile ester variations in Capsicum.

KEY MESSAGE: The transcript level of alcohol acyltransferase 1 (AAT1) may be the main factor influencing the variations in volatile esters that characterizing the fruity/exotic aroma of pepper fruit. Volatile esters are key components for characterizing the fruity/exotic aroma of pepper (Capsicum spp.) fruit. In general, the volatile ester content in the fruit is the consequence of a delicate balance between their synthesis by alcohol acyltransferases (AATs) and degradation by carboxylesterases (CXEs). However, the precise role of these families of enzymes with regard to volatile ester content remains unexplored in Capsicum. In this study, we found that the volatile ester content was relatively low in C. annuum and much higher in C. chinense, particularly in pungent varieties. Additionally, fruits collected from multiple non-pungent C. chinense varieties, which harbor loss-of-function mutations in capsaicinoid biosynthetic genes, acyltransferase (Pun1), putative aminotransferase (pAMT), or putative ketoacyl-ACP reductase (CaKR1) were analyzed. The volatile ester contents of non-pungent C. chinense varieties (pamt/pamt) were equivalent to those of pungent varieties, but their levels were significantly lower in non-pungent NMCA30036 (pun12/pun12) and C. chinense (Cakr1/Cakr1) varieties. Multiple AAT-like sequences were identified from the pepper genome sequences, whereas only one CXE-like sequence was identified. Among these, AAT1, AAT2, and CXE1 were isolated from fruits of C. chinense and C. annuum. Gene expression analysis revealed that the AAT1 transcript level is a potential determinant of fruit volatile ester variations in Capsicum. Furthermore, enzymatic assays demonstrated that AAT1 is responsible for the biosynthesis of volatile esters in pepper fruit. Identification of a key gene for aroma biosynthesis in pepper fruit will provide a theoretical basis for the development of molecular tools for flavor improvement.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum).

KEY MESSAGE: The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313 kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reduced normally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

Hyper-mobile Water and Raman 2900 cm-1 Peak Band of Water Observed around Backbone Phosphates of Double Stranded DNA by High-Resolution Spectroscopies and MD Structural Feature Analysis of Water.

High-resolution measurements of microwave dielectric relaxation and Raman spectroscopies for waters in double-stranded (ds) 10-mer DNA solution revealed the presence of hyper-mobile water (HMW) and a marked OH stretching band appearing in the range from 2500 to 3100 cm-1, here called the LA band, at the low wavenumber tail of the major OH stretching band of water. Quantitation of the Raman scattering intensity for ds 10-mer DNA in phosphate or tris(hydroxymethyl)aminomethane (TRIS) buffers showed that the LA band was formed by 2000-3000 water molecules per ds 10-mer DNA, indicating collective OH stretching vibrations of water molecules around the backbone phosphate oxygen atoms. The LA band intensity of ds 10-mer DNA in 10 mM TRIS increased and decreased by 30% with the addition of 2 mM MgCl2 and 2 mM CaCl2, respectively. The LA band intensity and the effect of adding Mg(II) or Ca(II) ions to the band intensity were maintained in the presence of 0.14 M KCl; however, the changes induced by the divalent cations were reduced by half. Molecular dynamics calculations of water molecules around the backbone phosphate groups of ds 10-mer DNA indicate the presence of high-density water and broad regions of fluctuating water density, suggesting that they correspond to HMW and the LA band, respectively.

A phase I, randomized, placebo-controlled study of molnupiravir in healthy Japanese to support special approval in Japan to treat COVID-19.

Molnupiravir (MK-4482) is an oral prodrug of the antiviral ribonucleoside analog, N-hydroxycytidine (NHC), which has activity against RNA viruses, including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). We conducted a phase I safety and pharmacokinetic study of molnupiravir in healthy Japanese adult participants. A sample size larger than typically used in pharmacokinetic studies was implemented to collect additional safety data in the Japanese population to support special approval for emergency use in Japan. Single doses of molnupiravir up to 1600 mg and multiple doses of 400 and 800 mg administered every 12 h (q12h) for 5.5 days were generally well-tolerated. NHC appeared rapidly in plasma and reached maximum concentration (Cmax ), with a median time to Cmax (Tmax ) between 1.00 and 2.00 h. Area under the concentration versus time curve from zero to infinity (AUC0-inf ), area under the concentration versus time curve from zero to 12 h (AUC0-12 ), and Cmax of plasma NHC increased approximately dose proportionally. With q12h dosing, the geometric mean (GM) accumulation ratios for NHC AUC0-12 and Cmax were ~1 for 400 and 800 mg. Pharmacokinetics of NHC triphosphate (NHC-TP), the active metabolite of NHC was assessed in peripheral blood mononuclear cells and also demonstrated roughly dose proportional pharmacokinetics. The GM accumulation ratios for NHC-TP AUC0-12 and Cmax were ~2.5 for 400 and 800 mg. Following administration with food, only a modest reduction (24%) in plasma NHC Cmax with comparable AUC0-inf was seen, supporting administration without regard to food.

Vanillin reduction in the biosynthetic pathway of capsiate, a non-pungent component of Capsicum fruits, is catalyzed by cinnamyl alcohol dehydrogenase.

Capsicum fruits synthesize capsaicin from vanillylamine, which is produced from vanillin in a reaction catalyzed by a putative aminotransferase (pAMT). Capsiate, a non-pungent compound that is structurally similar to capsaicin, is synthesized from vanillyl alcohol rather than vanillylamine. Vanillyl alcohol is possibly generated by the enzymatic reduction of vanillin, but the enzyme responsible for this reaction is unknown. In the present study, we revealed that the vanillin reductase in the capsiate biosynthetic pathway is cinnamyl alcohol dehydrogenase (CAD), which is an enzyme involved in lignin synthesis. The reduction of vanillin to vanillyl alcohol was greater in the mature red fruit placental extract than in the immature green fruit placental extract. This reduction was suppressed by both N-(O-hydroxyphenyl) sulfinamoyltertiobutyl acetate, a specific inhibitor of CAD, and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The CaCAD1 transcript levels in the placenta were higher in the red fruits than in the green fruits. A recombinant CaCAD1 protein obtained using an Escherichia coli expression system reduced vanillin to vanillyl alcohol. This reaction was suppressed by the CAD inhibitors. These results strongly suggest that CAD is the enzyme that catalyzes the reduction of vanillin to vanillyl alcohol during capsiate biosynthesis. Syntenic analyses indicated that genes encoding CAD and capsaicin synthase (Pun1) involved in capsiate biosynthesis were acquired before the pAMT gene during the evolution of the family Solanaceae. This raises the possibility that in the genus Capsicum, the capsiate biosynthetic pathway emerged before the pAMT-encoding gene was acquired as the final trigger for capsaicin biosynthesis.

Pathogenic changes in group 2 innate lymphoid cells (ILC2s) in a steroid-insensitive asthma model of mice.

A certain population of asthma patients is resistant to steroid therapy, whereas the mechanisms remain unclear. One of characteristic features of steroid-resistant asthma patients is severe airway eosinophilia based on type-2 inflammation. Aims of this study were: 1) to develop a murine model of steroid-resistant asthma, 2) to elucidate that predominant cellular source of a type-2 cytokine, IL-5 was group 2 innate lymphoid cells (ILC2s), 3) to analyze pathogenic alteration of ILC2s in the severe asthma, and 4) to evaluate therapeutic potential of anti-IL-5 monoclonal antibody (mAb) on the steroid-resistant asthma. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 5 or 500 µg/animal 4 times. Development of airway eosinophilia and remodeling in 5-µg OVA model were significantly suppressed by 1 mg/kg dexamethasone, whereas those in 500-µg OVA model were relatively insensitive to the dose of dexamethasone. ILC2s isolated from the lung of the steroid-insensitive model (500-µg OVA) produced significantly larger amounts of IL-5 in response to IL-33/TSLP than ILC2s from the steroid-sensitive model (5-µg OVA). Interestingly, TSLP receptor expression on ILC2s was up-regulated in the steroid-insensitive model. Treatment with anti-IL-5 mAb in combination with dexamethasone significantly suppressed the airway remodeling of the steroid-insensitive model. In conclusion, multiple intratracheal administration of a high dose of antigen induced steroid-insensitive asthma in sensitized mice. IL-5 was mainly produced from ILC2s, phenotype of which had been pathogenically altered probably through the up-regulation of TSLP receptors. IL-5 blockage could be a useful therapeutic strategy for steroid-resistant asthma.

Morphological and gene expression characterization of maf-1, a floral chili pepper mutant caused by a nonsense mutation in CaLFY.

Chili peppers are important as vegetables and ornamental crops, because of the variety of fruit shapes and colors. Understanding of flower and fruit development in Capsicum is limited compared with closely related Solanaceae crops such as tomato. This study reports a novel malformed fruit mutant named malformed fruit-1 (maf-1), which was isolated from an ethyl methanesulfonate-induced mutant population of chili pepper. maf-1 exhibited homeotic changes in the floral bud, which were characterized by conversion of petals and stamens into sepal-like and carpel-like organs, respectively. In addition, the indeterminate formation of carpel-like tissue was observed. Genetic analysis demonstrated that the causative gene in maf-1 is a nonsense mutation in CaLFY. This is the first characterization of an lfy mutant in Capsicum. Unlike tomatoes, the CaLFY mutation did not affect the architecture of sympodial unit or flowering time but mainly affected the formation of flower organs. Gene expression analysis suggested that a nonsense mutation in CaLFY led to decreased expression of multiple class B genes, resulting in homeotic changes in the flower and fruit. This maf-1 mutant may provide new insights at the molecular level in understanding flower organ formation and the genetic manipulation of fruit shape in chili peppers. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01304-w.

Synthesis of a Cyclic Dinucleotide Analogue with Ambiguous Bases, 5-Aminoimidazole-4-carboxamide.

Cyclic dinucleotides (CDNs) are second messengers composed of two purine nucleotides. In recent years, the structural diversity of CDNs and their functionality in biological processes are being intensely studied. Herein we report the chemical synthesis of cyclic di-5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl monophosphate (c-di-ZMP) (1), which consists of two 5-amino-4-imidazolecarboxamide ribonucleotides (Z-ribonucleotides) linked via two phosphodiester linkages. Construction of the CDN skeleton with an N1-dinitrophenylhypoxanthine base (HxaDNP-base) by phosphoramidite chemistry and the subsequent ring-opening reaction of HxaDNP-base successfully yielded the desired 1.

Capsaicinoid biosynthesis in the pericarp of chili pepper fruits is associated with a placental septum-like transcriptome profile and tissue structure.

KEY MESSAGE: CAP biosynthesis in the pericarp of chili pepper fruits occurs with an ambiguous boundary in the placental septum and pericarp. Capsaicinoid (CAP) is a pungent ingredient of chili pepper fruits. Generally, CAP biosynthesis is limited to the placental septum of fruits, but it has been reported that its biosynthesis occurs even in the pericarp of some extremely pungent varieties, resulting in a substantial increase in total content. To examine the mechanism of CAP biosynthesis in the pericarp, comparative transcriptome analysis of a variety that produces CAP in the pericarp (MY) and a variety that does not (HB) was carried out. RNA-seq revealed that 2264 genes were differentially expressed in the MY pericarp compared with the HB pericarp. PCA analysis and GO enrichment analysis indicated that the MY pericarp has a gene expression profile more like placental septum than the HB pericarp. The gene expression of CAP biosynthesis-related genes in the MY pericarp changed coordinately with the placental septum during fruit development. In most Capsicum accessions including HB, the distribution of slender epidermal cells producing CAP was limited to the placental septum, and the morphological boundary between the placental septum and pericarp was clear. In some extremely pungent varieties such as MY, slender epidermal cells ranged from the placental septum to the pericarp region, and the pericarp was morphologically similar to the placental septum, such as the absence of large sub-epidermal cells and abundant spaces in the parenchymal tissue. Our data suggest that CAP biosynthesis in the pericarp occurred with an ambiguous boundary in the placental septum and pericarp. These findings contribute to further enhancement of CAP production in chili pepper fruits.

Effect of cytosine-Ag+-cytosine base pairing on the redox potential of the Ag+/Ag couple and the chemical reduction of Ag+ to Ag by tetrathiafulvalene.

The redox properties of metallo-base pairs remain to be elucidated. Herein, we report the detailed 1H/13C/109Ag NMR spectroscopic and cyclic voltammetric characterisation of the [Ag(cytidine)2]+ complex as isolated cytosine-Ag+-cytosine (C-Ag+-C) base pairs. We also performed comparative studies between cytidine/Ag+ and other nucleoside/Ag+ systems by using cyclic voltammetry measurements. In addition, to evaluate the effect of [Ag(cytidine)2]+ formation on the chemical reduction of Ag+ to Ag, we utilised the redox reaction between Ag+ and tetrathiafulvalene (TTF). We found that Ag+-mediated base pairing lowers the redox potential of the Ag+/Ag couple. In addition, C-Ag+-C base pairing makes it more difficult to reduce captured Ag+ ions than in other nucleoside/Ag+ systems. Remarkably, the cytidine/Ag+ system can be utilised to control the redox potential of the Ag+/Ag couple in DMSO. This feature of the cytidine/Ag+ system may be exploited for Ag nanoparticle synthesis by using the redox reaction between Ag+ and TTF.

Data on swan arrival, departure, and population size on the Asadokoro tidal flat, Aomori Prefecture, Japan, from 1956 to 2010.

The arrival and departure dates and the daily maximum populations of migrating swans (Cygnus cygnus) on the Asadokoro tidal flat, Hiranai town, Aomori Prefecture, Japan, were recorded by elementary school students for more than 50 years between 1956 and 2010. The Asadokoro tidal flat, which lies along the coast of Mutsu Bay, has been designated a National Special Natural Monument, known as "The swans of Kominato and their migration grounds." This long history of observation unfortunately came to an end with the closure of the elementary school in 2012. If analyzed together with data on environmental factors, such as temperature changes or the effects of avian influenza, this dataset could provide a potentially valuable source of information, and consequently, future secondary use of the data is anticipated.

Chemical reduction of Ag+ to Ag employing organic electron donors: evaluation of the effect of Ag+-mediated cytosine-cytosine base pairing on the aggregation of Ag nanoparticles.

Ag+-mediated base pairing is valuable for synthesising DNA-based silver nanoparticles (AgNPs) and nanoclusters (AgNCs). Recently, we reported the formation of a [Ag(cytidine)2]+ complex in dimethyl sulfoxide (DMSO), which facilitated the evaluation of the effect of cytosine-Ag+-cytosine (C-Ag+-C) base pairing on the degree of AgNP aggregation in solution. As an aprotic solvent, DMSO was expected to dissolve the [Ag(cytidine)2]+ complex, and powerful reducing agents, such as organic electron donors. In this study, the chemical reduction of a cytidine/Ag+ system using a powerful reducing agent tetrakis(dimethylamino)ethylene (TDAE) was investigated. 1H/13C/15N NMR spectroscopic evidence was obtained to identify the iminium dication (TDAE2+), which is an oxidised form of TDAE. The results were compared with those obtained using another organic electron donor, tetrathiafulvalene (TTF), which exhibits a relatively lower reduction activity than TDAE. AgNPs prepared via redox reaction between [Ag(cytidine)2]+ and organic electron donors (TDAE and TTF) were characterised using UV-Vis spectroscopy and nanoparticle tracking analysis. It was found that the formation of C-Ag+-C base pairing inhibited the aggregation of AgNPs in solution. In addition, in the presence of cytidine, the total concentration of the AgNP solution was affected by the reduction activity of the reducing agent.

Post-hoc analysis of injection-site reactions following vaccination with quadrivalent human papillomavirus vaccine in Japanese female clinical trial participants.

AIM: The quadrivalent human papillomavirus (4vHPV) vaccine has demonstrated efficacy and immunogenicity and was generally well tolerated in clinical trials conducted in Japan. We report a detailed safety analysis of injection-site reactions in female Japanese 4vHPV clinical trial participants. METHODS: This post-hoc analysis included data from 2 double-blind, placebo-controlled phase II clinical trials of a 3-dose (Day 1, Month 2, Month 6) regimen of 4vHPV vaccine in Japanese young women aged 18-26 years (N = 1021; NCT00378560) and girls aged 9-17 years (N = 107; NCT00411749). Injection-site and systemic adverse events (AEs) were monitored using vaccination report cards for 15 days after each vaccine dose; serious AEs were reported throughout the trials. Post-hoc analyses of data from these trials were performed to examine details of injection-site AEs, including day of onset, time from onset to resolution, and maximum intensity. RESULTS: Injection-site AEs were reported by 85.6% of 4vHPV vaccine recipients and 72.4% of placebo recipients, most commonly erythema, pain, pruritus, and swelling (each >5% of 4vHPV vaccine recipients). The majority of injection-site AEs had an onset within 3 days of vaccination and were mild to moderate in intensity; few 4vHPV vaccine recipients reported severe injection-site AEs (2.0% overall). All injection-site AEs resolved, and most (4vHPV: 87.5%; placebo: 92.7%) resolved within 5 days of onset. CONCLUSIONS: Most injection-site reactions are mild or moderate in intensity and of short duration. The 3-dose regimen of 4vHPV vaccine is well tolerated in Japanese female clinical trial participants based on this post-hoc analysis. These results will further support safety communication between healthcare providers and vaccine recipients regarding the HPV vaccine. TRIAL REGISTRATION: Clinicaltrials. gov: NCT00378560 and NCT00411749.

Electrical conductance measurement of HgII-mediated DNA duplex in buffered aqueous solution.

Electrical properties of metal-mediated DNA duplexes (metallo-DNA) have been of particular interest because of their potential applications in DNA-based nanoelectronics. We prepared HgII-mediated DNA duplex with NH2 anchors and measured the electrical conductance of single-molecule metallo-DNA via scanning tunneling microscopy-based break junction method in the buffered solution. Three conductance values were observed that may correspond to different conformations of the metallo-DNA molecule bridged over metallic electrodes.

QM and QM/MM umbrella sampling MD study of the formation of Hg(II)-thymine bond: Model for evaluation of the reaction energy profiles in solutions with constant pH.

The formation of the Hg-N3(T) bond between the 1-methylthymine (T) molecule and the hydrated Hg2+ cation was explored with the combined quantum mechanics/molecular mechanics (QM/MM) method including Free Energy Perturbation corrections. The thermodynamic properties were determined in the whole pH range, when these systems were explicitly investigated and considered as the QM part: (1) T + [Hg(H2 O)6 ]2+ , (2) T + [Hg(H2 O)5 (OH)]+ , (3) T + Hg(H2 O)4 (OH)2 , and (4) N3-deprotonated T + Hg(H2 O)4 (OH)2 . The MM part contained only solvent molecules and counterions. As a result, the dependence of Gibbs-Alberty reaction free energy on pH was obtained along the reaction coordinate. We found that an endoergic reaction in acidic condition up to pH < 4-5 becomes exoergic for a higher pH corresponding to neutral and basic solutions. The migration of the Hg2+ cation between N3 and O4/2 positions in dependence on pH is discussed as well. For the verification, DFT calculations of stationary points were performed confirming the qualitative trends of QM/MM MD simulations and NMR parameters were determined for them.

The Wakayama-Immediate Stroke Life Support Course: Achieving Successful Training on the Stroke Emergency System without using Mechanical Medical Training Simulators.

BACKGROUND: The Immediate Stroke Life Support (ISLS) course run in Wakayama (Wakayama-ISLS course) is an off-the-job training course for understanding the initial treatment of acute stroke. A total of 22 Wakayama-ISLS courses have been held in Wakayama Prefecture since 2008. To begin with, the case presentation was performed using human-like, mechanical manikins for simulation training. However, as the course progressed, we found the students paying great attention to the display monitor, and less to the patients' neurological status. METHODS: From the fourth Wakayama-ISLS course onward, we conducted the group work with the facilitators pretending to be patients, i.e., without medical training manikins. RESULTS: When the facilitators acted as patients, the students gained a more realistic and expressive perception of neurological symptoms. As a result, they expressed a high level of satisfaction with the course in the questionnaire sent immediately afterwards, regardless of their profession or prior experience. Moreover, as we did not need to transfer medical training simulators, we were able to carry out three ISLS courses at locations some distance from Wakayama city on a low-cost basis. This also enabled the regional medical staff in rural hospitals to participate in the courses easily. CONCLUSION: The Wakayama-ISLS course without medical training manikins is an entirely feasible off-the-job training course, which provides training on fast and excellent treatment of acute stroke problems based on clinical practice. The course has the potential to spread not only across Japan but throughout the world, including to developing countries, given the cost perspective.

Structure-activity relationship of pseudoknot-type hammerhead ribozyme reveals key structural elements for enhanced catalytic activity†.

The pseudoknot-type hammerhead ribozyme (PK-HHRz) is known to be activated by a pseudoknot interaction between loops I and II. To obtain maximal activation through the pseudoknot formation, we studied the structure-activity relationship of PK-HHRz. From these studies, the structural requirements of the PK-HHRz cleavage reaction were clearly defined. In addition, we discovered a PK-HHRz with higher cleavage activity than the wild-type sequence. Although modifications generally disrupt the activity of enzymes, in this case the elongation of loop II increased the activity of PK-HHRz. These new findings will form a structural basis for designing PK-HHRz variants for gene-therapeutic/manipulating agents and biochemical/nanotechnological tools.