Evaluation of radiation dose and image quality for dental cone-beam computed tomography in pediatric patients.

Children are sensitive to radiation; therefore, it is necessary to reduce radiation dose as much as possible in pediatric patients. In addition, it is crucial to investigate the optimal imaging conditions as they considerably affect the radiation dose. In this study, we investigated the effect of different imaging conditions on image quality and optimized the imaging conditions for dental cone-beam computed tomography (CBCT) examinations to diagnose ectopic eruptions and impacted teeth in children. To achieve our aims, we evaluated radiation doses and subjective and objective image quality. The CBCT scans were performed using 3D Accuitomo F17. All combinations of a tube voltage (90 kV), tube currents (1, 2, 3 mA), fields of view (FOVs) (4 × 4, 6 × 6 cm), and rotation angles (360°, 180°) were used. Dose-area product values were measured. SedentexCT IQ cylindrical phantom was used to physically evaluate the image quality. We used the modulation transfer function as an index of resolution, the noise power spectrum as an index of noise characteristics, and the system performance function as an overall evaluation index of the image. Five dentists visually evaluated the images from the head-neck phantom. The results showed that the image quality tended to worsen, and scores for visual evaluation decreased as tube currents, FOVs and rotation angles decreased. In particular, image noise negatively affected the delineation of the periodontal ligament space. The optimal imaging conditions were 90 kV, 2 mA, 4 × 4 cm FOV and 180° rotation. These results suggest that CBCT radiation doses can be significantly reduced by optimizing the imaging conditions.

Impact of diagnosis and treatment of uterine fibroids on quality of life and labor productivity: The Japanese online survey for uterine fibroids and quality of life (JOYFUL survey).

AIM: To investigate the impact of uterine fibroid diagnosis/treatment status on quality of life (QOL) and work productivity in women living in Japan. METHODS: Women aged 20-49 years who registered on Macromill were recruited via the opt-in method. They completed an online survey on demographic and uterine fibroid diagnosis/treatment status, 36-Item Short-Form Health Survey, Uterine Fibroid Symptom and Health-Related Quality of Life questionnaire (UFS-QOL), and World Health Organization Health and Work Performance Questionnaire. RESULTS: There were 4120 respondents: 1362 untreated, 249 with ongoing treatment, 449 with past treatment, 1030 with no uterine fibroids, and 1030 with unknown uterine fibroid status. A high proportion of women with ongoing treatment had moderate to severe uterine fibroid-like symptoms (symptom severity score of UFS-QOL ≥40 points), accompanied by reduced QOL. QOL was improved in women with past treatment. Uterine fibroids had a significant impact on physical and psychosocial aspects in the ongoing treatment group versus other groups. Using classification and regression tree analysis, anemia was identified as a plausible predictor of reduced QOL in the ongoing treatment group. Approximately 20% of women-even in groups other than the ongoing treatment group-experienced moderate to severe uterine fibroid-like symptoms. However, the diagnosis and treatment status of uterine fibroids had no clear impact on work productivity. CONCLUSIONS: Uterine fibroids, especially in association with anemia, were related to reduced QOL. Given that uterine fibroid-related reduced QOL is likely improved by appropriate treatment, women with uterine fibroid-like symptoms, such as menorrhagia, should be examined and treated.

Evaluation of a New Real-Time Dosimeter Sensor for Interventional Radiology Staff.

In 2011, the International Commission on Radiological Protection (ICRP) recommended a significant reduction in the lens-equivalent radiation dose limit, thus from an average of 150 to 20 mSv/year over 5 years. In recent years, the occupational dose has been rising with the increased sophistication of interventional radiology (IVR); management of IVR staff radiation doses has become more important, making real-time radiation monitoring of such staff desirable. Recently, the i3 real-time occupational exposure monitoring system (based on RaySafeTM) has replaced the conventional i2 system. Here, we compared the i2 and i3 systems in terms of sensitivity (batch uniformity), tube-voltage dependency, dose linearity, dose-rate dependency, and angle dependency. The sensitivity difference (batch uniformity) was approximately 5%, and the tube-voltage dependency was <±20% between 50 and 110 kV. Dose linearity was good (R2 = 1.00); a slight dose-rate dependency (~20%) was evident at very high dose rates (250 mGy/h). The i3 dosimeter showed better performance for the lower radiation detection limit compared with the i2 system. The horizontal and vertical angle dependencies of i3 were superior to those of i2. Thus, i3 sensitivity was higher over a wider angle range compared with i2, aiding the measurement of scattered radiation. Unlike the i2 sensor, the influence of backscattered radiation (i.e., radiation from an angle of 180°) was negligible. Therefore, the i3 system may be more appropriate in areas affected by backscatter. In the future, i3 will facilitate real-time dosimetry and dose management during IVR and other applications.

Relugolix, an oral gonadotropin-releasing hormone receptor antagonist, reduces endometriosis-associated pain compared with leuprorelin in Japanese women: a phase 3, randomized, double-blind, noninferiority study.

OBJECTIVE: To evaluate the efficacy and safety of 40-mg relugolix (REL) compared with those of leuprorelin (LEU) in women with endometriosis-associated pain. DESIGN: Phase 3, multicenter, randomized, double-blind, double-dummy, active-controlled study in Japanese patients. SETTING: Hospitals and clinics. PATIENT(S): Women aged ≥20 years with regular menstrual cycles (25-38 days) experiencing endometriosis or ovarian endometrioma and reporting pelvic pain. INTERVENTION(S): In the REL group, 40 mg of REL was orally administered once a day for 24 weeks. In the LEU group, 3.75 or 1.88 mg of LEU was subcutaneously injected every 4 weeks for 24 weeks. MAIN OUTCOME MEASURE(S): The primary endpoint was the change in the maximum visual analog scale score for pelvic pain from baseline until 28 days before the end of treatment. RESULT(S): Changes in the maximum visual analog scale score were -52.6 ± 1.3 for REL and -57.5 ± 1.4 for LEU. Ovarian endometrioma decreased by 12.26 ± 17.52 cm3 for REL and 14.10 ± 18.81 cm3 for LEU. Drug-related treatment emergent adverse events with an incidence of >10% for both groups were hot flush, metrorrhagia, headache, and genital hemorrhage. Discontinuations from treatment emergent adverse events were 2.9% for REL and 4.3% for LEU. CONCLUSION(S): Relugolix was noninferior to LEU for treating endometriosis-associated pelvic pain. Safety profiles of both medications were comparable, although menses returned earlier in patients taking REL, a huge benefit for women who plan to conceive after treatment. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT03931915.

Association between serum albumin and cognitive dysfunction in hepatic encephalopathy: An exploratory data analysis.

BACKGROUND AND AIM: Because covert hepatic encephalopathy (CHE) has been shown to affect the prognosis of cirrhotic patients, early diagnosis of hepatic encephalopathy (HE) is a prerequisite for the preservation of patients' quality of life and for prophylaxis of overt HE. The aim of this study was to identify a clinical parameter to predict impairment of cognitive function in cirrhotic patients with early-stage HE. METHODS: We investigated the data from 172 patients with cirrhotic or idiopathic portosystemic shunt (PSS) in phase II/III trials of rifaximin in Japan. Classification and regression trees (CARTs) were constructed to identify clinical profiles related to cognitive dysfunction as indicated by the prolongation of time required for the Number Connection Test (NCT-B). RESULTS: CART analysis detected age 65 years as the variable for the initial split, and serum albumin level was selected as the variable for the second split among patients aged ≤65 years. In 27 cirrhotic patients aged ≤65 years without PSS, receiver operating characteristic curve analysis revealed that the optimal albumin level cutoff point was 3.05 g/dL, and the area under the curve was 0.80 for the prolongation of NCT-B time, which was higher than that of the branched-chain amino acids-to-tyrosine ratio (0.46), the prothrombin time-international normalized ratio (PT-INR) (0.68), serum ammonia (0.61), and total bilirubin (0.69). CONCLUSIONS: Lower serum albumin level as a clinical biomarker associated with impaired cognitive function may be available as a screening examination for early-stage HE in cirrhotic patients aged ≤65 years without PSS before undergoing neuropsychological tests.

Rifaximin-altered gut microbiota components associated with liver/neuropsychological functions in patients with hepatic encephalopathy: An exploratory data analysis of phase II/III clinical trials.

AIMS: Rifaximin (RFX), a non-systemic antibiotic, improves liver/neuropsychological functions in patients with hepatic encephalopathy (HE). We aimed to investigate the clinical profiles associated with gut bacterial loads using exploratory data analysis and the effects of RFX on the gut microbiota of patients with HE. METHODS: We analyzed the data from 17 patients with HE who underwent fecal microbiota examination in phase II/III trials in Japan. Profiles associated with genera Streptococcus, Veillonella, and Lactobacillus loads were analyzed using classification and regression trees (CART). Changes in gut microbial consortia of seven patients with HE were then assessed 2 weeks after RFX treatment by principal component analysis. RESULTS: In the CART, the first and second divergence variables for each higher bacterial load were as follows: (i) in Streptococcus, the number connection test-A ≥39.55 s and presence of portal-systemic shunt; (ii) in Veillonella, serum potassium levels <4.75 mEq/L and total cholesterol level <129.5 mg/dL; and (iii) in Lactobacillus, white blood cell counts ≥3.4 × 103 /µL and aspartate aminotransferase level ≥44.5 U/L. There was no significant change in total bacterial load before and after RFX treatment; however, there was a decrease in Streptococcus, Veillonella, and Lactobacillus counts after RFX treatment. CONCLUSION: We report clinical profiles associated with gut bacterial loads in patients with HE, and showed that RFX altered gut microbiota components associated with liver/neuropsychological functions. Thus, RFX could improve liver/neuropsychological functions through the regulation of the gut microbial consortia in patients with HE.

Tomato FRUITFULL homologs regulate fruit ripening via ethylene biosynthesis.

Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.

The AP2/ERF transcription factor SlERF52 functions in flower pedicel abscission in tomato.

In plants, abscission removes senescent, injured, infected, or dispensable organs. Induced by auxin depletion and an ethylene burst, abscission requires pronounced changes in gene expression, including genes for cell separation enzymes and regulators of signal transduction and transcription. However, the understanding of the molecular basis of this regulation remains incomplete. To examine gene regulation in abscission, this study examined an ERF family transcription factor, tomato (Solanum lycopersicum) ETHYLENE-RESPONSIVE FACTOR 52 (SlERF52). SlERF52 is specifically expressed in pedicel abscission zones (AZs) and SlERF52 expression is suppressed in plants with impaired function of MACROCALYX and JOINTLESS, which regulate pedicel AZ development. RNA interference was used to knock down SlERF52 expression to show that SlERF52 functions in flower pedicel abscission. When treated with an abscission-inducing stimulus, the SlERF52-suppressed plants showed a significant delay in flower abscission compared with wild type. They also showed reduced upregulation of the genes for the abscission-associated enzymes cellulase and polygalacturonase. SlERF52 suppression also affected gene expression before the abscission stimulus, inhibiting the expression of pedicel AZ-specific transcription factor genes, such as the tomato WUSCHEL homologue, GOBLET, and Lateral suppressor, which may regulate meristematic activities in pedicel AZs. These results suggest that SlERF52 plays a pivotal role in transcriptional regulation in pedicel AZs at both pre-abscission and abscission stages.

Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins.

The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN.

The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.

Expression profiling of tomato pre-abscission pedicels provides insights into abscission zone properties including competence to respond to abscission signals.

BACKGROUND: Detachment of plant organs occurs in abscission zones (AZs). During plant growth, the AZ forms, but does not develop further until the cells perceive abscission-promoting signals and initiate detachment. Upon signal perception, abscission initiates immediately; if there is no signal, abscission is not induced and the organ remains attached to the plant. However, little attention has been paid to the genes that maintain competence to respond to the abscission signal in the pre-abscission AZ. Recently, we found that the tomato (Solanum lycopersicum) transcription factors BLIND (Bl), GOBLET (GOB), Lateral suppressor (Ls) and a tomato WUSCHEL homologue (LeWUS) are expressed specifically in pre-abscission tissue, the anthesis pedicel AZs. To advance our understanding of abscission, here we profiled genome-wide gene expression in tomato flower pedicels at the pre-abscission stage. RESULTS: We examined the transcriptomes of three tomato flower pedicel regions, the AZ and flanking proximal- (Prox) and distal- (Dis) regions, and identified 89 genes that were preferentially expressed in the AZ compared to both Prox and Dis. These genes included several transcription factors that regulate apical or axillary shoot meristem activity. Also, genes associated with auxin activity were regulated in a Prox-Dis region-specific manner, suggesting that a gradient of auxin exists in the pedicel. A MADS-box gene affecting floral transition was preferentially expressed in the Prox region and other MADS-box genes for floral organ identification were preferentially expressed in Dis, implying that the morphologically similar Prox and Dis regions have distinct identities. We also analyzed the expression of known regulators; in anthesis pedicels, Bl, GOB, Ls and LeWUS were expressed in the vascular cells of the AZ region. However, after an abscission signal, Bl was up-regulated, but GOB, Ls and LeWUS were down-regulated, suggesting that Bl may be a positive regulator of abscission, but the others may be negative regulators. CONCLUSIONS: This study reveals region-specific gene expression in tomato flower pedicels at anthesis and identifies factors that may determine the physiological properties of the pre-abscission pedicel. The region-specific transcriptional regulators and genes for auxin activity identified here may prevent flower abscission in the absence of signal or establish competence to respond to the abscission signal.

The regulatory mechanism of fruit ripening revealed by analyses of direct targets of the tomato MADS-box transcription factor RIPENING INHIBITOR.

The developmental process of ripening is unique to fleshy fruits and a key factor in fruit quality. The tomato (Solanum lycopersicum) MADS-box transcription factor RIPENING INHIBITOR (RIN), one of the earliest-acting ripening regulators, is required for broad aspects of ripening, including ethylene-dependent and -independent pathways. However, our knowledge of direct RIN target genes has been limited, considering the broad effects of RIN on ripening. In a recent work published in The Plant Cell, we identified 241 direct RIN target genes by chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) and transcriptome analysis. Functional classification of the targets revealed that RIN participates in the regulation of many biological processes including well-known ripening processes such as climacteric ethylene production and lycopene accumulation. In addition, we found that ethylene is required for the full expression of RIN and several RIN-targeting transcription factor genes at the ripening stage. Here, based on our recently published findings and additional data, we discuss the ripening processes regulated by RIN and the interplay between RIN and ethylene.

A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening.

The fruit ripening developmental program is specific to plants bearing fleshy fruits and dramatically changes fruit characteristics, including color, aroma, and texture. The tomato (Solanum lycopersicum) MADS box transcription factor RIPENING INHIBITOR (RIN), one of the earliest acting ripening regulators, is required for both ethylene-dependent and -independent ripening regulatory pathways. Recent studies have identified two dozen direct RIN targets, but many more RIN targets remain to be identified. Here, we report the large-scale identification of direct RIN targets by chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) targeting the predicted promoters of tomato genes. Our combined ChIP-chip and transcriptome analysis identified 241 direct RIN target genes that contain a RIN binding site and exhibit RIN-dependent positive or negative regulation during fruit ripening, suggesting that RIN has both activator and repressor roles. Examination of the predicted functions of RIN targets revealed that RIN participates in the regulation of lycopene accumulation, ethylene production, chlorophyll degradation, and many other physiological processes. Analysis of the effect of ethylene using 1-methylcyclopropene revealed that the positively regulated subset of RIN targets includes ethylene-sensitive and -insensitive transcription factors. Intriguingly, ethylene is involved in the upregulation of RIN expression during ripening. These results suggest that tomato fruit ripening is regulated by the interaction between RIN and ethylene signaling.

Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses.

The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.

MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development.

Abscission in plants is a crucial process used to shed organs such as leaves, flowers, and fruits when they are senescent, damaged, or mature. Abscission occurs at predetermined positions called abscission zones (AZs). Although the regulation of fruit abscission is essential for agriculture, the developmental mechanisms remain unclear. Here, we describe a novel transcription factor regulating the development of tomato (Solanum lycopersicum) pedicel AZs. We found that the development of tomato pedicel AZs requires the gene MACROCALYX (MC), which was previously identified as a sepal size regulator and encodes a MADS-box transcription factor. MC has significant sequence similarity to Arabidopsis (Arabidopsis thaliana) FRUITFULL, which is involved in the regulation of fruit dehiscent zone development. The MC protein interacted physically with another MADS-box protein, JOINTLESS, which is known as a regulator of fruit abscission; the resulting heterodimer acquired a specific DNA-binding activity. Transcriptome analyses of pedicels at the preabscission stage revealed that the expression of the genes involved in phytohormone-related functions, cell wall modifications, fatty acid metabolism, and transcription factors is regulated by MC and JOINTLESS. The regulated genes include homologs of Arabidopsis WUSCHEL, REGULATOR OF AXILLARY MERISTEMS, CUP-SHAPED COTYLEDON, and LATERAL SUPPRESSOR. These Arabidopsis genes encode well-characterized transcription factors regulating meristem maintenance, axillary meristem development, and boundary formation in plant tissues. The tomato homologs were specifically expressed in AZs but not in other pedicel tissues, suggesting that these transcription factors may play key roles in pedicel AZ development.

Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation.

BACKGROUND: During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. RESULTS: Using immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. CONCLUSIONS: The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own promoter suggests the presence of autoregulation for RIN expression. ChIP-based analyses identified a novel RIN-binding CArG-box site that harbors a gene associated with RIN expression in its flanking region. These findings clarify the crucial role of RIN in the transcriptional regulation of ripening initiation and progression.

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis.

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene-enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous-nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome-wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri-nucleotide repeats were most common among the SSRs and were overrepresented in the protein-coding sequences, we found that selection against indels of tri-nucleotide repeats was relatively weak in both African and Asian rice. Our genome-wide sequencing of O. glaberrima and in-depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.