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ImportanceQuality of life (QOL) of patients with atopic dermatitis (AD) is reported to be the lowest among skin diseases. To our knowledge, mindfulness and self-compassion training has not been evaluated for adults with AD.ObjectiveTo evaluate the efficacy of mindfulness and self-compassion training in improving the QOL for adults with AD.Design, Setting, and ParticipantsThis randomized clinical trial conducted from March 2019 through October 2022 included adults with AD whose Dermatology Life Quality Index (DLQI) score, a skin disease–specific QOL measure, was greater than 6 (corresponding to moderate or greater impairment). Participants were recruited from multiple outpatient institutes in Japan and through the study’s social media outlets and website.InterventionsParticipants were randomized 1:1 to receive eight 90-minute weekly group sessions of online mindfulness and self-compassion training or to a waiting list. Both groups were allowed to receive any dermatologic treatment except dupilumab.Main Outcomes and MeasuresThe primary outcome was the change in the DLQI score from baseline to week 13. Secondary outcomes included eczema severity, itch- and scratching-related visual analog scales, self-compassion and all of its subscales, mindfulness, psychological symptoms, and participants’ adherence to dermatologist-advised treatments.ResultsThe study randomized 107 adults to the intervention group (n = 56) or the waiting list (n = 51). The overall participant mean (SD) age was 36.3 (10.5) years, 85 (79.4%) were women, and the mean (SD) AD duration was 26.6 (11.7) years. Among participants from the intervention group, 55 (98.2%) attended 6 or more of the 8 sessions, and 105 of all participants (98.1%) completed the assessment at 13 weeks. The intervention group demonstrated greater improvement in the DLQI score at 13 weeks (between-group difference estimate, −6.34; 95% CI, −8.27 to −4.41; P d) at 13 weeks was −1.06 (95% CI, −1.39 to −0.74). All secondary outcomes showed greater improvements in the intervention group than in the waiting list group.Conclusions and RelevanceIn this randomized clinical trial of adults with AD, integrated online mindfulness and self-compassion training in addition to usual care resulted in greater improvement in skin disease–specific QOL and other patient-reported outcomes, including eczema severity. These findings suggest that mindfulness and self-compassion training is an effective treatment option for adults with AD.Trial Registrationhttps://umin.ac.jp/ctr Identifier: UMIN000036277

To what extent the COVID-19 pandemic and its containment measures influenced mental health in the general population is still unclear.To assess the trajectory of mental health symptoms during the first year of the pandemic and examine dose-response relations with characteristics of the pandemic and its containment.Relevant articles were identified from the living evidence database of the COVID-19 Open Access Project, which indexes COVID-19-related publications from MEDLINE via PubMed, Embase via Ovid, and PsycInfo. Preprint publications were not considered.Longitudinal studies that reported data on the general population's mental health using validated scales and that were published before 31 March 2021 were eligible.An international crowd of 109 trained reviewers screened references and extracted study characteristics, participant characteristics, and symptom scores at each timepoint. Data were also included for the following country-specific variables: days since the first case of SARS-CoV-2 infection, the stringency of governmental containment measures, and the cumulative numbers of cases and deaths.In a total of 43 studies (331 628 participants), changes in symptoms of psychological distress, sleep disturbances, and mental well-being varied substantially across studies. On average, depression and anxiety symptoms worsened in the first 2 months of the pandemic (standardized mean difference at 60 days, -0.39 [95% credible interval, -0.76 to -0.03]); thereafter, the trajectories were heterogeneous. There was a linear association of worsening depression and anxiety with increasing numbers of reported cases of SARS-CoV-2 infection and increasing stringency in governmental measures. Gender, age, country, deprivation, inequalities, risk of bias, and study design did not modify these associations.The certainty of the evidence was low because of the high risk of bias in included studies and the large amount of heterogeneity. Stringency measures and surges in cases were strongly correlated and changed over time. The observed associations should not be interpreted as causal relationships.Although an initial increase in average symptoms of depression and anxiety and an association between higher numbers of reported cases and more stringent measures were found, changes in mental health symptoms varied substantially across studies after the first 2 months of the pandemic. This suggests that different populations responded differently to the psychological stress generated by the pandemic and its containment measures.Swiss National Science Foundation. (PROSPERO: CRD42020180049).

Major depression is often a relapsing disorder. It is therefore important to start its treatment with therapies that maximize the chance of not only getting the patients well but also keeping them well. We examined the associations between initial treatments and sustained response by conducting a network meta-analysis of randomized controlled trials (RCTs) in which adult patients with major depression were randomized to acute treatment with a psychotherapy (PSY), a protocolized antidepressant pharmacotherapy (PHA), their combination (COM), standard treatment in primary or secondary care (STD), or pill placebo, and were then followed up through a maintenance phase. By design, acute phase treatment could be continued into the maintenance phase, switched to another treatment or followed by discretionary treatment. We included 81 RCTs, with 13,722 participants. Sustained response was defined as responding to the acute treatment and subsequently having no depressive relapse through the maintenance phase (mean duration: 42.2±16.2 weeks, range 24-104 weeks). We extracted the data reported at the time point closest to 12 months. COM resulted in more sustained response than PHA, both when these treatments were continued into the maintenance phase (OR=2.52, 95% CI: 1.66-3.85) and when they were followed by discretionary treatment (OR=1.80, 95% CI: 1.21-2.67). The same applied to COM in comparison with STD (OR=2.90, 95% CI: 1.68-5.01 when COM was continued into the maintenance phase; OR=1.97, 95% CI: 1.51-2.58 when COM was followed by discretionary treatment). PSY also kept the patients well more often than PHA, both when these treatments were continued into the maintenance phase (OR=1.53, 95% CI: 1.00-2.35) and when they were followed by discretionary treatment (OR=1.66, 95% CI: 1.13-2.44). The same applied to PSY compared with STD (OR=1.76, 95% CI: 0.97-3.21 when PSY was continued into the maintenance phase; OR=1.83, 95% CI: 1.20-2.78 when PSY was followed by discretionary treatment). Given the average sustained response rate of 29% on STD, the advantages of PSY or COM over PHA or STD translated into risk differences ranging from 12 to 16 percentage points. We conclude that PSY and COM have more enduring effects than PHA. Clinical guidelines on the initial treatment choice for depression may need to be updated accordingly.

ObjectiveAlthough mindfulness and self-compassion programs have been applied in clinical settings, no studies have reported their benefits for atopic dermatitis (AD). The aim of the study was to evaluate the feasibility and acceptability of an online group format of 8 weekly 90-minute sessions of a mindfulness and self-compassion program for adults with AD.ResultsIn this single-arm, open label pre-post comparison study design, 15 adults (10 women; mean age, 38.3 ± 8.98 years) completed the intervention and assessment. The primary outcome was disease-specific quality of life (QOL) (Dermatology Life Quality Index and Skindex-16) at the 20-week follow-up. The secondary outcomes were other self-reported questionnaires, and descriptive feedback of the program. Assessments were conducted at baseline, mid-treatment, post-intervention, and at follow-up. Overall, there were no significant improvements. However, of the four participants whose QOL was largely impacted due to AD, the scores improved significantly, with minimal clinically important difference or greater change. All participants were satisfied with the program. This program could benefit adults with AD by improving QOL. Randomized controlled trials are warranted to elucidate the efficacy of the program. This study has been registered at the UMIN Clinical Trials Registry (ID: UMIN000030484).

AimsThe Hamilton Depression Rating Scale (HAMD) and the Beck Depression Inventory (BDI) are the most frequently used observer-rated and self-report scales of depression, respectively. It is important to know what a given total score or a change score from baseline on one scale means in relation to the other scale.MethodsWe obtained individual participant data from the randomised controlled trials of psychological and pharmacological treatments for major depressive disorders. We then identified corresponding scores of the HAMD and the BDI (369 patients from seven trials) or the BDI-II (683 patients from another seven trials) using the equipercentile linking method.ResultsThe HAMD total scores of 10, 20 and 30 corresponded approximately with the BDI scores of 10, 27 and 42 or with the BDI-II scores of 13, 32 and 50. The HAMD change scores of −20 and −10 with the BDI of −29 and −15 and with the BDI-II of −35 and −16.ConclusionsThe results can help clinicians interpret the HAMD or BDI scores of their patients in a more versatile manner and also help clinicians and researchers evaluate such scores reported in the literature or the database, when scores on only one of these scales are provided. We present a conversion table for future research.

OBJECTIVES: Control conditions' influence on effect estimates of active psychotherapeutic interventions for depression has not been fully elucidated. We used network meta-analysis to estimate the differences between control conditions.STUDY DESIGN AND SETTING: We have conducted a comprehensive literature search of randomized trials of psychotherapies for adults with depression up to January 1, 2019 in four major databases (PubMed, PsycINFO, Embase, and Cochrane). The network meta-analysis included broadly conceived cognitive behavior therapies in comparison with the following control conditions: Waiting List (WL), No Treatment (NT), Pill Placebo (PillPlacebo), Psychological Placebo (PsycholPlacebo).RESULTS: 123 studies with 12,596 participants were included. The I-squared was 55.9% (95% CI: 45.9%; to 64.0%) (moderate heterogeneity). The design-by-treatment global test of inconsistency was not significant (P = 0.44). Different control conditions led to different estimates of efficacy for the same intervention. WL appears to be the weakest control (odds ratio of response against NT = 1.93 (1.30 to 2.86), PsycholPlacebo = 2.03 (1.21 to 3.39), and PillPlacebo = 2.66 (1.45 to 4.89), respectively).CONCLUSIONS: Different control conditions produce different effect estimates in psychotherapy randomized controlled trials for depression. WL was the weakest, followed by NT, PsycholPlacebo, and PillPlacebo in this order. When conducting meta-analyses of psychotherapy trials, different control conditions should not be lumped into a single group.

During development, the fruit of some paprika (Capsicum annuum L.) cultivars shows a change in color from green to dark purple (e.g., ‘Mavras’) or lilac (e.g., ‘Tequila’). However, this purple coloration is rare among paprika cultivars and disappears in ripened fruit, which are red. Therefore, we investigated the mechanism causing this color change in the cultivars Mavras and Tequila to better understand how purple ripened fruit could be generated. High-performance liquid chromatography (HPLC) analyses of the anthocyanin contents of the fruit indicated that anthocyanin was undetectable in green fruit, accumulated in dark purple or lilac ones, and then decreased again in red ones in both cultivars. Furthermore, expressions of most of the analyzed anthocyanin biosynthesis–related genes and genes for their transcription factors increased in dark purple or lilac fruit and decreased in red ones, i.e., it was synchronized with the changes in anthocyanin contents. Furthermore, anthocyanin degradation activity as a result of peroxidases was detected at all stages but increased when the lilac or dark purple color started to fade. Thus, the development of purple coloration is caused by increased anthocyanin biosynthesis, whereas the fading of this coloration is a result of both a decrease in anthocyanin biosynthesis and an increase in anthocyanin degradation. At the ripening stage, the green pigment (chlorophyll) contents decreased, whereas the red pigment (carotenoid, particularly capsanthin) contents increased. However, these timings did not completely coincide with the timing of anthocyanin degradation, suggesting that the content of each pigment is individually regulated, and so purple, green, and red coloration could be freely expressed in mature paprika fruit.

IntroductionWe have recently compared all second-generation as well as selected first-generation antidepressants in terms of efficacy and acceptability in the acute treatment of major depression. Here we present a protocol for a network meta-analysis aimed at extending these results, updating the evidence base and comparing all second-generation as well as selected first-generation antidepressants in terms of specific adverse events and tolerability in the acute treatment of major depression in adults.Methods and analysisWe will include all double-blind randomised controlled trials comparing one active drug with another or with placebo in the acute treatment major depression in adults. We will compare the following active agents: agomelatine, amitriptyline, bupropion, citalopram, clomipramine, desvenlafaxine, duloxetine, escitalopram, fluoxetine, fluvoxamine, levomilnacipran, milnacipran, mirtazapine, nefazodone, paroxetine, reboxetine, sertraline, trazodone, venlafaxine, vilazodone and vortioxetine. The main outcomes will include the total number of patients experiencing specific adverse events; experiencing serious adverse events; and experiencing at least one adverse event. Published and unpublished studies will be retrieved through relevant database searches, trial registries and websites; reference selection and data extraction will be completed by at least two independent reviewers. For each outcome we will undertake a network meta-analysis to synthesise all evidence. We will use local and global methods to evaluate consistency. We will perform all analyses in R. We will assess the quality of evidence contributing to network estimates with the Confidence in Network Meta-Analysis web application.DiscussionThis work will provide an in- depth analysis and an insight into the specific adverse events of individual antidepressants.Ethics and disseminationThis review does not require ethical approval.PROSPERO registration numberCRD42019128141.

We have previously found that a gene closely related to Arabidopsis CONSTANS-like 16 (COL16) was coordinately expressed with chlorophyll content in chrysanthemum petals and leaves. Here, to elucidate whether COL16 is involved in the regulation of chlorophyll biosynthesis and accumulation, we analyzed the function of COL16 in petunia (Petunia hybrida). We identified three petunia COL16 homologs: PhCOL16a, PhCOL16b, and PhCOL16c. Expression patterns of all three homologs were associated with chlorophyll content, with lower levels in white corollas than in pale green corollas, and relatively high levels in leaves. The result suggests that PhCOL16 homologs are involved in chlorophyll accumulation. We introduced a PhCOL16a overexpression construct into petunia. The transgenic plants had pale green corollas with a higher chlorophyll content than wild-type plants. Expression of genes encoding key enzymes of chlorophyll biosynthesis was significantly higher in the transgenic plants than in the wild-type plants. The results indicate that PhCOL16 positively regulates chlorophyll biosynthesis.

Petunia (Petunia hybrida) is an important ornamental plant with a wide range of corolla colors. Although pale-yellow-flowered cultivars, with a low amount of carotenoids in their corollas, are now available, no deep-yellow-flowered cultivars exist. To find why petunia cannot accumulate enough carotenoids to have deep-yellow flowers, we compared carotenoid profiles and expression of carotenoid metabolic genes between pale-yellow-flowered petunia and deep-yellow-flowered calibrachoa (Calibrachoa hybrida), a close relative. The carotenoid contents and the ratios of esterified xanthophylls to total xanthophylls in petunia corollas were significantly lower than those in calibrachoa, despite similar carotenoid components. A lower esterification rate of trans-xanthophylls than of cis-xanthophylls in petunia suggests that petunia xanthophyll esterase (XES) has low substrate specificity for trans-xanthophylls, which are more abundant than cis-xanthophylls in petunia corolla. The expression of genes encoding key enzymes of carotenoid biosynthesis was lower and that of a carotenoid catabolic gene was higher in petunia. XES expression was significantly lower in petunia. The results suggest that low biosynthetic activity, high cleavage activity, and low esterification activity cause low carotenoid accumulation in petunia corollas.

Objectives Drug-resistant overactive bladder (OAB) represents an unmet medical need in that treatment options are limited. We developed a treatment model based on cognitive behavioral therapy and evaluated its feasibility and acceptability for drug-resistant OAB in women. Methods This was an open-label, single-arm, multicenter pilot study. We defined drug-resistant OAB as OAB with moderate to severe symptoms despite pharmacotherapy for more than 12 weeks. A face-to-face intervention was prescribed as six sessions (30 minutes each) over 6 to 12 weeks according to a treatment manual. The effects were assessed by self-reported questionnaires and frequency voiding charts (FVC) at baseline, during intervention, immediately after intervention, and at follow-up. Results Ten patients participated in this study. Median age was 72 years, median OAB Symptom Score was nine points, and median duration of prior treatment for OAB was 5.5 years at baseline. Two participants dropped out of the study. Among the remaining patients, the scores of the OAB Questionnaire subscales improved (effect size: 0.75-1.73), and the mean urinary frequency in the FVC also improved from baseline (9.0 times, SD: 2.1) to follow-up (6.2 times, SD: 1.2). All participants were satisfied with the intervention. There were no adverse events during this study. Conclusions The new treatment based on cognitive behavioral therapy was well tolerated and feasible in women with drug-resistant OAB. Further randomized research is needed to rigorously evaluate the efficacy of the treatment.

Carnation is one of the most important floricultural crops in the world. Over 20 wild species of Dianthus have been integrated during the long history of carnation breeding, and a wide range of petal colors, including red, yellow, orange, and white, have been developed. These flower colors are derived from quantitative and qualitative differences in flavonoid compounds. Wild Dianthus species expressing petal colors through carotenoids or chlorophylls have not been documented. In contrast, green-flowered and pale yellow-flowered carnation cultivars, which express their petal colors with these pigments, have been developed by conventional breeding. Such cultivars might be mutants that have lost the ability to avoid the accumulation of carotenoids and chlorophylls in the petals. In this section, based on our analysis of green- and pale yellow-flowered carnation cultivars, we discuss the possible mechanisms that regulate the accumulation of carotenoids and chlorophylls in carnation petals.

Petunia (Petunia hybrida) is an important ornamental plant, with corolla colors ranging widely from pink to red to purple, owing mainly to anthocyanins. Although there are no bright-yellow-flowered cultivars, some pale-yellow-flowered cultivars accumulate a small amount of carotenoids. To find a key regulatory step that controls carotenoid content in petunia corollas, we compared the expression of carotenoid metabolism genes and carotenoid composition in corollas of white-flowered and pale-yellow-flowered cultivars. Pale yellow corollas tended to have higher expression of biosynthesis genes. The most prominent result was the complete lack of carotenoid cleavage dioxygenase 4a (CCD4a) transcripts in pale yellow corollas. We found two insertions, one in the putative promoter region and the other in the coding region, of the genomic CCD4a sequence of a pale-yellow-flowered cultivar relative to that of a white-flowered cultivar. We consider this the main reason for the lack of CCD4a transcripts. The results suggest that pale yellow corollas have higher carotenoid biosynthesis activity and lower catabolism activity than white corollas. We propose that carotenoid content in petunia corollas is determined by the balance of the degradation and biosynthesis of carotenoids.

The color variations of ornamental flowers are often generated by ion-beam and gamma irradiation mutagenesis. However, mutation rates differ significantly even among cultivars of the same species, resulting in high cost and intensive labor for flower color breeding. We aimed to establish a metabolome-based strategy to identify biomarkers and select promising parental lines with high mutation rates using Chrysanthemum as the case study. The mutation rates associated with flower color were measured in 10 chrysanthemum cultivars with pink, yellow, or white flowers after soft X-ray irradiation at the floret-formation stage. The metabolic profiles of the petals of these cultivars were clarified by widely targeted metabolomics and targeted carotenoid analysis using liquid chromatography-tandem quadrupole mass spectrometry. Metabolome and carotenoid data were subjected to an un-supervised principal component analysis (PCA) and a supervised logistic regression with least absolute shrinkage and selection operator (LASSO). The PCA of the metabolic profile data separated chrysanthemum cultivars according to flower color rather than mutation rates. By contrast, logistic regression with LASSO generated a discrimination model to separate cultivars into two groups with high or low mutation rates, and selected 11 metabolites associated with mutation rates that can be biomarkers candidates for selecting parental lines for mutagenesis. This metabolome-based strategy to identify metabolite markers for mutation rates associated with flower color might be applied to other ornamental flowers to accelerate mutation breeding for generating new cultivars with a wider range of flower colors.

The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.

IntroductionRecent advances in troponin sensitivity enabled early and accurate judgement of ruling-out myocardial infarction, especially non-ST elevation myocardial infarction (NSTEMI) in emergency departments (EDs) with development of various prediction-rules and high-sensitive-troponin-based strategies (hs-troponin). Reliance on clinical impression, however, is still common, and it remains unknown which of these strategies is superior. Therefore, our objective in this prospective cohort study is to comprehensively validate the diagnostic accuracy of clinical impression-based strategies, prediction-rules and hs-troponin-based strategies for ruling-out NSTEMIs.Methods and analysisIn total, 1500 consecutive adult patients with symptoms suggestive of acute coronary syndrome will be prospectively recruited from five EDs in two tertiary-level, two secondary-level community hospitals and one university hospital in Japan. The study has begun in July 2018, and recruitment period will be about 1 year. A board-certified emergency physician will complete standardised case report forms, and independently perform a clinical impression-based risk estimation of NSTEMI. Index strategies to be compared will include the clinical impression-based strategy; prediction rules and hs-troponin-based strategies for the following types of troponin (Roche Elecsys hs-troponin T; Abbott ARCHITECT hs-troponin I; Siemens ADVIA Centaur hs-troponin I; Siemens ADVIA Centaur sensitive-troponin I). The reference standard will be the composite of type 1 MI and cardiac death within 30 days after admission to the ED. Outcome measures will be negative predictive value, sensitivity and effectiveness, defined as the proportion of patients categorised as low risk for NSTEMI. We will also evaluate inter-rater reliability of the clinical impression-based risk estimation.Ethics and disseminationThe study is approved by the Ethics Committees of the Kyoto University Graduate School and Faculty of Medicine and of the five hospitals where we will recruit patients. We will disseminate the study results through conference presentations and peer-reviewed journals.

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3′,5′-hydroxylase gene (F3′5′H), which encodes the key enzyme for delphinidin biosynthesis. In F3′5′H -transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3′5′H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3′5′H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5′-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red–purple to a purple–violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

We evaluated a novel primary three-dimensional culture system for human hepatocytes using micro-space plates. The functional activity of human hepatocytes in primary culture was determined by measuring albumin secretion from hepatocytes to medium and measuring expression levels of albumin, CYP1A2 and CYP3A4 mRNA. Albumin secretion was higher in micro-space plates compared with traditional plates after 72 h of culture; the levels of albumin secretion from hepatocytes to medium in culture using micro-space plates after 96 h of culture were 2.7-fold higher than those in culture using traditional plates, and secretion of albumin in micro-space plate culture subsequently remained constant. Expression levels of albumin, CYP1A2 and CYP3A4 mRNA in the culture of hepatocytes were significantly higher using micro-space plates than using traditional plates. The inducibility of CYP1A2 and CYP3A4 mRNA after exposure to inducers in hepatocyte culture on micro-space plates was comparable to that in culture on traditional plates, while expression of CYP1A2 and CYP3A4 mRNA after exposure to inducers was higher on micro-space plates than on traditional plates. The present study demonstrates that a novel primary three-dimensional culture system of cryopreserved human hepatocytes using micro-space plates could be used for evaluating the induction of drug-metabolizing enzymes in humans. This in vitro method may thus be useful for screening the induction potency of new drug candidates.

Carotenoids are one of the most important pigments for petal coloration in the yellow to red range; however, knowledge of carotenoids in petals is relatively limited. To better understand flower carotenoids, we analyzed carotenoid composition, the expression of carotenogenic genes, and the relationship between pigment composition and petal color in petals of Compositae plants, including chrysanthemums. We found that petals of yellow-flowered chrysanthemums have a unique carotenoid composition, and that the formation of white petal color in chrysanthemums involves carotenoid degradation catalyzed by carotenoid cleavage dioxygenase (CmCCD4a). We also showed three routes to an orange petal color via carotenoid components in 9 Compositae plants. In addition, we identified (5Z)-carotenoids that contribute orange color formation in calendula petals. In this review, we summarize our studies on carotenoids in the petals of Compositae plants.

We analyzed the petals of orange- and yellow-flowered cultivars of 9 Compositae (Asteraceae) species for total anthocyanin content, total carotenoid content, and carotenoid composition to clarify the mechanisms responsible for the differences in color. Petals of both orange- and yellow-flowered cultivars of these species contained yellowish carotenoids in common. There were three different ways for orange-flowered cultivars to add redness to the yellow base in order to form orange petals. The first way to display orange is to accumulate more anthocyanins than yellow-flowered cultivars; orange petals of Chrysanthemum morifolium, Gerbera jamesonii, and Zinnia elegans are mainly formed in this way. The second is to accumulate more carotenoids than yellowflowered cultivars; orange petals of Helianthus annuus, Tagetes erecta, and Tagetes petula are formed in this way. The third is to accumulate more reddish carotenois than yellow-flowered cultivars; orange petals of Calendula officinalis, Gazania spp., and Osteospermum ecklonis are mainly formed in this way. These three ways could be combined through inbreeding to produce orange flowers that vary in color tones.

We analyzed carotenoid composition and the content and expression of genes encoding isoprenoid and carotenoid biosynthetic enzymes in petals and leaves of chrysanthemums. Most of the carotenoids in yellow petals were β,ɛ-carotenoids, lutein and its derivatives, reflecting the high expression levels of lycopene ɛ-cyclase (LCYE). In contrast, the ratio of β,β-carotenoids to total carotenoids in leaves were higher than that of β,ɛ-carotenoids, reflecting the high expression levels of lycopen β-cyclase (LCYB). Petals of the yellow-flowered cultivar Yellow Paragon showed increased accumulation and drastic componential changes of carotenoids as they matured. In petals of the white-flowered cultivar Paragon, carotenoid content was drastically decreased during petal development and became less than the detection limit late in development. Transcript levels of most genes tested increased during petal development in Yellow Paragon. All genes except that for 1-deoxyxylulose 5-phosphate synthase (DXS) showed similar expression patterns in Paragon. Between-cultivar comparison of the expression of these genes in the petals at mid-development showed no distinct differences between petal color. It is possible that the formation of white petal color is due to neither down-regulation nor destruction of the carotenoid biosynthetic pathway. We presume that another factor inhibits carotenoid accumulation in chrysanthemum petals.

We investigated the usefulness of the promoter of a gene for tobacco elongation factor 1α protein (EF1 α) for transgene expression in the chrysanthemum (Chrysanthemum morifolium Ramat.). The EF1 α promoter was fused to the β-glucuronidase gene (gus) and introduced into the chrysanthemum. We obtained 238 putative transformants and GUS assay of the leaves of the in vitro plants revealed that 29.0% (69/238) of the putative plants were GUS-positive. The plants in the greenhouse that were 20 months after regeneration still showed a GUS activity in their leaves and petals. The tobacco EF1 α promoter expressed the transgene more efficiently than the 35S promoter of Cauliflower mosaic virus and could be used for transgene expression in chrysanthemum.

Sixteen xanthophylls were isolated from the petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Among them, (3S,5S,6R,3'R,6'R)-5,6-dihydro-5,6-dihydroxylutein (1) and five di-Z geometrical isomers of lutein-5,6-epoxide, i.e., 9Z,13'Z (2), 13Z,9'Z (3), 9'Z,13'Z (4), 9Z,13Z (5), and 9Z,9'Z (7), had never before been identified as natural products. All of the carotenoids isolated from chrysanthemum, except for (9Z)-violaxanthin, are beta,epsilon-carotene (alpha-carotene) derivatives. The analyses indicate that carotenoids from the petals of chrysanthemum have a very characteristic composition.

Variations in chloroplast DNA of Dendranthema species were studied by PCR-RFLP analysis to seek the maternal origin of the cultivated chrysanthemum, D. grandlflorum. Ten genes, atpH, matK, petA, petB, psaA, rbcL, rpoE, rpoC, trnK and 76S in the chloroplast DNA of 12 Japanese wild species and 1 cultivar were amplified by PCR. The amplified DNAs that were digested with each of 32 restriction endonucleases revealed 13 site changes among the 13 species in the following 9 gene plus restriction endonuclease combinations: petA-Avall, petA-Haelll, petA-MboII, petA-NdeII, rpoC-EcoRV, trnK-Dral, trnK-HinfI, trnK MboII and trnK-ScrFI. No Japanese wild species showed the same PCR-RFLP pattern of chloroplast DNA as D. grandiflorum.

We report a method for Agrobacterium -mediated transformation of Elatior Begonia ( Begonia × hiemalis Fotsch). Young leaf discs were infected with Agrobacterium tumefaciens strains AGL0 and LBA4404. Each strain has a binary vector plasmid, pIG121Hm that includes the β -glucuronidase (GUS) gene with an intron as a reporter gene, and both the neomycin phosphotransferase II and the hygromycin phosphotransferase genes as selection markers. Explants were cultured on modified MS medium supplemented with 1.0 mg/l BA, 0.5 mg/l IAA, 300 mg/l ticarcillin, and either 100 mg/l kanamycin and 5 mg/l hygromycin, or 300 mg/l kanamycin for selection and regeneration. Out of 500 explants infected with AGL0, 16 plantlets were regenerated, and out of 628 explants infected with LBA4404, two plantlets were regenerated after 4 months of culture. Transformation was confirmed by Southern blot analysis of the GUS gene and by histochemical assays of GUS activity in plant tissues. Ten in vitro transgenic plants were obtained from AGL0 infected explants only.

Coexpression of two anthocyanin modification genes elicits blue flower coloration through interaction with colorless flavonoids., Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)–glucose:anthocyanin 3′,5′-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3′5′H. Newly synthesized 3′,5′-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed “copigmentation,” with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.

Modification of flower colour in torenia (Torenia fournieri Lind.) by reintroduction of the chalcone synthase (CHS) or dihydroflavonol-4-reductase genes has been reported (Aida et al., 2000.Plant Science153: 33–42). The typical modified phenotype among plants with an introduced antisense gene is a uniformly lighter-coloured corolla. Of the 67 plants in which an antisense CHS gene was introduced, only a single line (411-7) showed a wavy pattern on the flower lip. In flowers of this plant, the inner part of the corolla lip was pigmented more deeply than the outer part in a wave-like pattern—a pattern that does not exist in normal cultivars. The segregation ratio of the flower colour patterns of the offspring and Southern blot analysis demonstrated that one of the two transgene loci detected may cause the wavy phenotype; the other locus is never associated with the wavy phenotype but alone it could produce the typical antisense type pattern.

When anthocyanins in plants make complexes with copigments such as flavones or flavonols (copigmentation), the visible absorption maximum of the flowers is shifted so that it becomes longer: that is, the flowers look bluer. In an earlier study, our group reported the modification of flower color in torenia (Torenia fournieri Lind.) by re-introduction of the dihydroflavonol-4-reductase (DFR) gene or the chalcone synthase (CHS) gene. Our initial observation of torenia transformants was that plants with the antisense DFR gene produced bluer flowers than plants with the antisense CHS gene. In the present study we found that inactivation of the DFR gene by genetic transformation caused the accumulation of flavones - possible copigments - and that the resulting copigmentation likely to make the torenia flowers bluer. This method could be applied to other plant species to produce bluer flowers.

We modified flower color in torenia (Torenia fournieri Lind.) by transferring the chalcone synthase (CHS) or the dihydroflavonol-4-reductase (DFR) gene in sense or antisense orientation by Agrobacterium-mediated gene transfer. The modification patterns of flower color among the transformants formed three groups: (1) same color as the wild-type plant; (2) whole corolla changed to a uniformly light color; and (3) with greater degree of lightening in the tube than in the lip. Transformants incorporating antisense transgene(s) tended to become group 2 types, with no plants becoming group 3 type. Transformants harboring sense transgene(s) tended to become group 3 types, rather than group 2 types. Sense genes and antisense genes seemed to have different potential for changing the flower color. We also produced transformants with new characters in torenia flower color; for example, lines with pastel flowers, wavy patterned flowers and parti-colored flowers. We regard this system to be useful for flower color breeding in torenia and for studying gene expression.

Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5′ position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462–464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver. Furthermore, we show that the rate of change of expression levels of many target mRNAs relative to the value before inoculation of the hepatocytes into micro-space plates was relatively smaller than the rate of change in hepatocytes inoculated into traditional plates. Pharmacokinetics-related examinations using this system are possible within a time frame of 120 h. We report that this novel three-dimensional culture system reproduces mRNA expression levels that are nearer to those in the liver in vivo and is an excellent platform for maintaining mRNA expression levels of drug-metabolizing enzymes and transporters when compared to common monolayer cultures.

The development of professional identity for healthcare professionals in the modern era has included the expectation that one must set aside personal feelings and life experience in the interest of...

ARTCEREB irrigation and perfusion solution (Artcereb), an ethical pharmaceutical, is typically applied inside the skull and spinal cavity as artificial fluid. Artcereb is composed of glucose and electrolytes (Na+, K+, Mg2+, Ca2+, Cl-, HCO3- and P) and has a pH of 7.3. An in vitro assessment of the effects of Artcereb on cell culture of rat fetal astrocytes or rat fetal brain cells was performed in comparison with normal saline and lactated Ringer's solutions. Furthermore, the effects of Artcereb on cell culture of rat fetal brain cells were also assessed in comparison with Krebs bicarbonate solution. Cell function after exposure to Artcereb was assessed based on 3H-thymidine incorporation activity. Cell function after exposure to Artcereb and lactated Ringer's solution in primary cultures of rat fetal astrocytes remained unaffected when compared to that after exposure to normal saline. Cell function after exposure to Artcereb in a primary culture of rat brain cells remained unaffected as compared to that after exposure to normal saline and lactated Ringer's solution. However, function decreased after exposure to a modified Artcereb formulation lacking bicarbonate, thus confirming that the presence of bicarbonate is essential for the Artcereb formulation.

In an attempt to create an animal model of constipation in monkeys, amitriptyline was administered to cynomolgus monkeys at doses of 10-160 mg/kg body weight via a nasogastric tube. Normal control monkeys excreted feces frequently throughout the day. Monkeys treated with amitriptyline at doses of 10-40 mg/kg showed delays in feces excretion. The 60 mg/kg treated monkeys for the most part did not excrete feces during the 24 h after amitriptyline administration. The 80 and 120 mg/kg treated monkeys did not excrete feces until 24 h from administration of amitriptyline, and also showed prolonged crouching and lethargy. On the other hand, 160 mg/kg treated monkeys died within 24 h after administration. We therefore felt that the optimal dose for creating constipation in the monkeys was 60 mg/kg. We tested the appropriateness of this amitriptyline-induced constipated monkey model by observing the effects of a new laxative, the herbal medicine ND-10 and the commercially available laxative bisacodyl. Control monkeys (those not receiving ND-10 or bisacodyl) treated with 60 mg/kg amitriptyline did not excrete feces up to 32 h after amitriptyline administration in 2 of 3 monkeys. However, all monkeys treated with one tablet of ND-10 excreted feces. Also, in 4 monkeys administrated with bisacodyl, 3 excreted feces. In this study, we confirmed that constipation can be caused in cynomolgus monkeys by oral administration of amitriptyline. This model may also be useful for the evaluation of laxatives.

Japanese morning glory (Ipomoea nil) is a representative plant lacking a yellow-flowered cultivar, although a few wild Ipomoea species contain carotenoids in their petals such as Ipomoea sp. (yellow petals) and I. obscura (pale-yellow petals). In the present study, carotenoid composition and the expression patterns of carotenogenic genes during petal development were compared among I. nil, I. obscura, and Ipomoea sp. to identify the factors regulating carotenoid accumulation in Ipomoea plant petals. In the early stage, the carotenoid composition in petals of all the Ipomoea plants tested was the same as in the leaves mainly showing lutein, violaxanthin, and beta-carotene (chloroplast-type carotenoids). However, in fully opened flowers, chloroplast-type carotenoids were entirely absent in I. nil, whereas they were present in trace amounts in the free form in I. obscura. At the late stage of petal development in Ipomoea sp., the majority of carotenoids were beta-cryptoxanthin, zeaxanthin, and beta-carotene (chromoplast-type carotenoids). In addition, most of them were present in the esterified form. Carotenogenic gene expression was notably lower in I. nil than in Ipomoea sp. In particular, beta-ring hydroxylase (CHYB) was considerably suppressed in petals of both I. nil and I. obscura. The CHYB expression was found to be significantly high in the petals of Ipomoea sp. during the synthesis of chromoplast-type carotenoids. The expression levels of carotenoid cleavage genes (CCD1 and CCD4) were not correlated with the amount of carotenoids in petals. These results suggest that both I. obscura and I. nil lack the ability to synthesize chromoplast-type carotenoids because of the transcriptional down-regulation of carotenogenic genes. CHYB, an enzyme that catalyses the addition of a hydroxyl residue required for esterification, was found to be a key enzyme for the accumulation of chromoplast-type carotenoids in petals.

The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed polymerase chain reaction-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV-vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5'), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5'Z,9'Z)-lycopene (3), (5'Z)-gamma-carotene (4), and (5'Z,9'Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5'Z)-lycopene (2) has been reported only as a synthesized compound.

Changes in the levels of CYP4A1, PPARalpha, and RXRalpha mRNA expression in the liver following overdose of fat-free or fat-containing total parenteral nutrition (TPN) were studied in 3-week-old male Sprague-Dawley rats. The rats were divided into three groups: group 1, oral diet; group 2, fat-free TPN; and group 3, TPN with 20% of calories from soybean oil emulsion. Levels of CYP4A1, PPARalpha, and RXRalpha mRNA in the fat-free TPN group were significantly lower than those in the other groups. Levels of CYP4A1 and PPARalpha mRNA were strongly correlated (r=0.849), and levels of CYP4A1 and RXRalpha mRNA were weakly correlated (r=0.618). This is the first report of a strong correlation between the levels of CYP4A1 and PPARalpha mRNA following overdose of fat-free or fat-containing TPN in infant rats. Our results also indicate that it is important to include fat in TPN regimens in order to prevent CYP4A1 mRNA down-regulation, which may be related to changes in PPARalpha mRNA levels in the liver.

m s d a i e The presentation and explanation of data in Fig. 4 in the riginal publication, which included real-time PCR analysis f DG-ERS1 mRNA in ligulate corollas and leaves, could be isleading due to an error in calculation of mRNA levels n which the normalization of the copy number of DG-ERS1 RNA in respective samples was done against the mean copy umber of actin mRNA as a reference. We have re-examined he changes of DG-ERS1 mRNA levels in flower and leaf issues of the both cultivars and corrected the way the noralization was made. The expression ratio of the DG-ERS1