Preference of treatment characteristics among people with haemophilia or their caregivers, and physicians in the Japanese healthcare environment.

INTRODUCTION: Studies of treatment preferences in haemophilia have been conducted in many countries. This study is the first to examine treatment characteristic preferences among people with haemophilia (PWH) and their caregivers, and physicians in Japan. AIM: To examine current treatment preferences of PWH and their caregivers, plus those of physicians at haemophilia treatment centres (HTCs) and non-HTCs for different treatment characteristics in Japan. METHODS: Physicians listed on a survey panel were invited to participate in the survey and to refer PWH and caregivers to participate in the survey. Web-based surveys were conducted to examine physician and PWH/caregiver background, prophylaxis background, prophylaxis goals, understanding of haemophilia treatment products, important information sources, preferences while choosing prophylaxis products, understanding of the patient's condition, and potential product switching. A discrete choice experiment exercise was included in the survey. RESULTS: A total of 107 physicians and 44 PWH/caregivers participated in the study. Key treatment goals of physicians included optimisation of haemophilia management. PWH/caregivers were focused on quality of life and reduced treatment burden. Consistent differences in haemophilia treatment strategies at HTCs and non-HTCs were observed for prescribed treatments, preferences in choosing prophylaxis products, understanding of patients' condition, and reasons for potential product switch. CONCLUSION: Our study utilises real-world survey data and presents preferences for haemophilia treatment characteristics among physicians, PWH and their caregivers in Japan, which could encourage improvements in individualised treatment and disease management. Alignment between treatment approaches at HTCs and non-HTCs could facilitate improvements in the quality of care for PWH across Japan.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Visualization of phosphorus re-translocation and phosphate transporter expression profiles in a shortened annual cycle system of poplar.

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P are considered an important strategy to support the vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under 'a shortened annual cycle system', which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32 P and/or 33 P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.

Two pathways of 2n gamete formation and differences in the frequencies of 2n gametes between wild species and interspecific hybrids.

KEY MESSAGE: Epidendrum produces 2n gametes with high frequency. This paper is the first to report on multiple pathways for forming 2n gametes, meiotic defeats, and pre-meiotic chromosome doubling. Unreduced 2n reproductive cells are predominantly involved in pathways that lead to polyploid plants. Although one of the most common pathways for inducing 2n gametes is through meiotic defects, a small set of isolated species alternatively generates 2n gametes from tetraploid pollen mother cells in the pre-meiotic phase. Hence, determining the mechanisms underlying 2n gamete formation is critical to improving breeding programmes and understanding plant evolution. We investigated sporads to reveal the pathway(s) accounting for the formation and frequencies of 2n gametes in wild species and interspecific hybrids in the genus Epidendrum. We investigated different types of sporads with varying frequencies, sizes, and viability in the wild species and hybrids of the genus Epidendrum. Large tetrad-estimated pre-meiotic chromosome doubling was observed in wild species. The Epidendrum is unique in that it forms 2n pollens via two pathways, namely, meiotic defects and pre-meiotic chromosome doubling. These two pathways of 2n pollen formation could influence the high diversity generation of polyploidy with different degrees of heterozygosity and genetic backgrounds in the genus Epidendrum. Therefore, these findings are proposed to influence polyploid breeding of Epidendrum via 2n pollen, helping us understand evolution and speciation via unreduced 2n gamete formation in Orchidaceae.

Genomic Basis of Transcriptome Dynamics in Rice under Field Conditions.

How genetic variations affect gene expression dynamics of field-grown plants remains unclear. Expression quantitative trait loci (eQTL) analysis is frequently used to find genomic regions underlying gene expression polymorphisms. This approach requires transcriptome data for the complete set of the QTL mapping population under the given conditions. Therefore, only a limited range of environmental conditions is covered by a conventional eQTL analysis. We sampled sparse time series of field-grown rice from chromosome segment substitution lines (CSSLs) and conducted RNA sequencing (RNA-Seq). Then, by using statistical analysis integrating meteorological data and the RNA-Seq data, we identified 1,675 eQTLs leading to polymorphisms in expression dynamics under field conditions. A genomic region on chromosome 11 influences the expression of several defense-related genes in a time-of-day- and scaled-age-dependent manner. This includes the eQTLs that possibly influence the time-of-day- and scaled-age-dependent differences in the innate immunity between Koshihikari and Takanari. Based on the eQTL and meteorological data, we successfully predicted gene expression under environments different from training environments and in rice cultivars with more complex genotypes than the CSSLs. Our novel approach of eQTL identification facilitated the understanding of the genetic architecture of expression dynamics under field conditions, which is difficult to assess by conventional eQTL studies. The prediction of expression based on eQTLs and environmental information could contribute to the understanding of plant traits under diverse field conditions.

Auxin Contributes to the Intraorgan Regulation of Gene Expression in Response to Shade.

Plants sense and respond to light via multiple photoreceptors including phytochrome. The decreased ratio of red to far-red light that occurs under a canopy triggers shade-avoidance responses, which allow plants to compete with neighboring plants. The leaf acts as a photoperceptive organ in this response. In this study, we investigated how the shade stimulus is spatially processed within the cotyledon. We performed transcriptome analysis on microtissue samples collected from vascular and nonvascular regions of Arabidopsis (Arabidopsis thaliana) cotyledons. In addition, we mechanically isolated and analyzed the vascular tissue. More genes were up-regulated by the shade stimulus in vascular tissues than in mesophyll and epidermal tissues. The genes up-regulated in the vasculature were functionally divergent and included many auxin-responsive genes, suggesting that various physiological/developmental processes might be controlled by shade stimulus in the vasculature. We then investigated the spatial regulation of these genes in the vascular tissues. A small vascular region within a cotyledon was irradiated with far-red light, and the response was compared with that when the whole seedling was irradiated with far-red light. Most of the auxin-responsive genes were not fully induced by the local irradiation, suggesting that perception of the shade stimulus requires that a wider area be exposed to far-red light or that a certain position in the mesophyll and epidermis of the cotyledon be irradiated. This result was consistent with a previous report that auxin synthesis genes are up-regulated in the periphery of the cotyledon. Hence, auxin acts as an important intraorgan signaling factor that controls the vascular shade response within the cotyledon.

Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change.

MAIN CONCLUSION: Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active.

Real-world long-term safety and effectiveness of turoctocog alfa in the treatment of haemophilia A in Japan: results from a multicentre, non-interventional, post-marketing study.

OBJECTIVES: To assess the safety and effectiveness of turoctocog alfa in previously treated patients (PTPs) and previously untreated patients (PUPs) with haemophilia A in a real-world setting in Japan. METHODS: This multicentre, non-interventional, post-marketing study recruited patients with haemophilia A who initiated treatment with turoctocog alfa from 18 sites (08/2014-12/2018). The primary endpoint was adverse events (AEs) during the 2-year study period. RESULTS: The safety and effectiveness analysis set included 39 patients. In total, 13 (33.3%) patients reported ≥1 AE; incidence rate was 60.4 events/100 patient-years of exposure (PYE). Treatment was withdrawn in two cases: pruritus in a PTP and factor VIII inhibitor development in a PUP. Inhibitor development occurred in 2.6% of all patients, with an incidence rate of 3.8 events/100 PYE. The rate of inhibitor development was 0%, 25% and 20% in PTPs, PUPs and PUPs with severe type, respectively. The haemostatic success rate was 91.4% for 383 bleeding episodes and 85.7% for 14 surgeries. The negative binomial annualised bleeding rate for the prophylaxis regimen was 6.19 episodes/year (95% CI, 3.69-10.38). The mean (SD) total consumption of turoctocog alfa (n = 34; excluding FVIII inhibitors) was 5,382.6 (7,180.1) IU/kg/year/patient; consumption was 4,133.1 (1,452.4) IU/kg/year/patient for prophylaxis. DISCUSSION: The effectiveness and safety profiles were comparable to those observed in other turoctocog alfa trials; effectiveness analysis and consumption were not affected by treatment regimens. CONCLUSION: Long-term use of turoctocog alfa therapy in clinical practice posed no newly identified safety issues and was effective for prophylaxis and treatment of bleeds in patients with haemophilia A in Japan.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars.

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.

Effect of differences in light source environment on transcriptome of leaf lettuce (Lactuca sativa L.) to optimize cultivation conditions.

When used in closed-type plant factories, light-emitting diode (LED) illumination systems have the particular advantages of low heat emission and high luminous efficiency. The effects of illumination quality and intensity on the growth and morphogenesis of many plant species have been examined, but improvements are needed to optimize the illumination systems for better plant products with lower resource investments. In particular, new strategies are needed to reduce the wastage of plant products related to leaf senescence, and to better control the ingredients and appearance of leafy vegetables. Although the quality of light is often altered to change the characteristics of plant products, the transcriptional status underlying the physiological responses of plants to light has not been established. Herein, we performed a comprehensive gene expression analysis using RNA-sequencing to determine how red, blue, and red/blue LEDs and fluorescent light sources affect transcriptome involved in the leaf aging of leaf lettuce. The RNA-sequencing profiling revealed clear differences in the transcriptome between young and old leaves. Red LED light caused large variation between the two age classes, while a pure or mixed blue LED light spectrum induced fewer transcriptome differences between young and old leaves. Collectively, the expression levels of genes that showed homology with those of other model organisms provide a detailed physiological overview, incorporating such characteristics as the senescence, nutrient deficiency, and anthocyanin synthesis of the leaf lettuce plants. Our findings suggest that transcriptome profiles of leaf lettuce grown under different light sources provide helpful information to achieve better growth conditions for marketable and efficient green-vegetable production, with improved wastage control and efficient nutrient inputs.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Induction of adventitious shoots and tetraploids in Antirrhinum majus L. by treatment of antimitotic agents in vitro without plant growth regulators.

We examined the effects of five antimitotic agents using Antirrhinum majus L. 'Maryland True Pink' on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.

Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms.

RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine sequencers. In order to reduce cost, we used cost-effective and robust commercial regents with small reaction volumes. This method is a whole mRNA-Seq, which can be applied even to non-model organisms lacking the transcriptome references. In addition, we designed large number of 3' PCR primer including 8 nucleotides barcode sequences for multiplexing up to three hundreds samples. To summarize, it is possible with this protocol to prepare 96 directional RNA-Seq libraries from purified total RNA in three days and can be pooled for up to three hundred libraries. This is beneficial for large scale transcriptome analysis in many fields of animals and plant biology.

Transcriptional Variation in Glucosinolate Biosynthetic Genes and Inducible Responses to Aphid Herbivory on Field-Grown Arabidopsis thaliana.

Recently, increasing attempts have been made to understand how plant genes function in natura. In this context, transcriptional profiles represent plant physiological status in response to environmental stimuli. Herein, we combined high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana grown at a field site in Switzerland. We found that genes with the gene ontology (GO) annotations of "glucosinolate biosynthetic process" and "response to insects" were most significantly enriched, and the expression of these genes was highly variable among plant accessions. Nearly half of the total expression variation in the glucosinolate biosynthetic genes (AOPs, ESM1, ESP, and TGG1) was explained by among-accession variation. Of these genes, the expression level of AOP3 differed among Col-0 accession individuals depending on the abundance of the mustard aphid (Lipaphis erysimi). We also found that the expression of the major cis-jasmone activated gene CYP81D11 was positively correlated with the number of flea beetles (Phyllotreta striolata and Phyllotreta atra). Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to attack by mustard aphids. The combined results from RNA-Seq and our ecological survey illustrate the feasibility of using field transcriptomics to detect an inducible defense, providing a first step towards an in natura understanding of biotic interactions involving phenotypic plasticity.

Histogen Layers Contributing to Adventitious Bud Formation Are Determined by their Cell Division Activities.

Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars "Kaname," "Concord," and "Monique" were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3', 5' hydroxylase (SiF3'5'H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using "Kaname," all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used "Concrd" and "Monique" from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of "Kaname." On the other hand, in "Concord" and "Monique," the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from "Concord" and "Monique" were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity.