Self-incompatibility phenotypes of SRK mutants can be predicted with high accuracy.

Only very limited information is available on why some non-synonymous variants severely alter gene function while others have no effect. To identify the characteristic features of mutations that strongly influence gene function, this study focused on S-locus receptor kinase, SRK, which encodes a highly polymorphic receptor kinase expressed in stigma papillary cells that underlies a female determinant of self-incompatibility in Brassicaceae. A set of 299 Arabidopsis thaliana transformants expressing mutated SRKb from A. lyrata was constructed and analyzed to determine the genotype and self-incompatibility phenotype of each transformant. Almost all the transformants showing the self-incompatibility defect contained mutations in AlSRKb that altered localization to the plasma membrane. The observed mutations occurred in amino acid residues that were highly conserved across S haplotypes and whose predicted locations were in the interior of the protein. These mutations were likely to underlie the self-incompatibility defect as they caused significant changes to amino acid properties. Such findings suggested that mutations causing the self-incompatibility defect were more likely to result from changes to AlSRKb biosynthesis than from loss of function. In addition, this study showed the RandomForest and Extreme Gradient Boosting methods could predict self-incompatibility phenotypes of SRK mutants with high accuracy.

Prednisolone improves hippocampal regeneration after trimethyltin-induced neurodegeneration in association with prevention of T lymphocyte infiltration.

The endogenous regenerative capacity of the brain is quite weak; however, a regenerative reaction, the production of new neurons (neurogenesis), has been reported to occur in brain lesions. In addition, leukocytes are well known to infiltrate brain lesions. Therefore, leukocytes would also have a link with regenerative neurogenesis; however, their role has not been fully elucidated. In this study, we investigated leukocyte infiltration and its influence on brain tissue regeneration in a trimethyltin (TMT)-injected mouse model of hippocampal regeneration. Immunohistochemically, CD3-positive T lymphocytes were found in the hippocampal lesion of TMT-injected mice. Prednisolone (PSL) treatment inhibited T lymphocyte infiltration and increased neuronal nuclei (NeuN)-positive mature neurons and doublecortin (DCX)-positive immature neurons in the hippocampus. Investigation of bromodeoxyuridine (BrdU)-labeled newborn cells revealed the percentage of BrdU/NeuN- and BrdU/DCX-positive cells increased by PSL treatment. These results indicate that infiltrated T lymphocytes prevent brain tissue regeneration by inhibiting hippocampal neurogenesis.

Epistatic interactions among multiple copies of FLC genes with naturally occurring insertions correlate with flowering time variation in radish.

Brassicaceae crops, which underwent whole-genome triplication during their evolution, have multiple copies of flowering-related genes. Interactions among multiple gene copies may be involved in flowering time regulation; however, this mechanism is poorly understood. In this study, we performed comprehensive, high-throughput RNA sequencing analysis to identify candidate genes involved in the extremely late-bolting (LB) trait in radish. Then, we examined the regulatory roles and interactions of radish FLOWERING LOCUS C (RsFLC) paralogs, the main flowering repressor candidates. Seven flowering integrator genes, five vernalization genes, nine photoperiodic/circadian clock genes and eight genes from other flowering pathways were differentially expressed in the early-bolting (EB) cultivar 'Aokubinagafuto' and LB radish cultivar 'Tokinashi' under different vernalization conditions. In the LB cultivar, RsFLC1 and RsFLC2 expression levels were maintained after 40 days of cold exposure. Bolting time was significantly correlated with the expression rates of RsFLC1 and RsFLC2. Using the EB × LB F2 population, we performed association analyses of genotypes with or without 1910- and 1627-bp insertions in the first introns of RsFLC1 and RsFLC2, respectively. The insertion alleles prevented the repression of their respective FLC genes under cold conditions. Interestingly, genotypes homozygous for RsFLC2 insertion alleles maintained high RsFLC1 and RsFLC3 expression levels under cold conditions, and two-way analysis of variance revealed that RsFLC1 and RsFLC3 expression was influenced by the RsFLC2 genotype. Our results indicate that insertions in the first introns of RsFLC1 and RsFLC2 contribute to the late-flowering trait in radish via different mechanisms. The RsFLC2 insertion allele conferred a strong delay in bolting by inhibiting the repression of all three RsFLC genes, suggesting that radish flowering time is determined by epistatic interactions among multiple FLC gene copies.

Short-Term Intake of Chlorogenic Acids Improves Psychomotor Speed and Motor Speed in Adults: A Randomized Crossover Trial.

Chlorogenic acid (CGA), a polyphenolic compound found in various plants, has been reported to improve cognitive function. However, it remains unclear how long it takes for CGAs to exert their effects. Here, we evaluated the short-term effects of CGAs on cognitive function. We assessed the effects of 2-week CGA intake on cognitive function. The study was carried out as a randomized, double-blind, placebo-controlled, crossover trial. Twenty-six healthy Japanese participants (50-65 years of age) were randomly assigned to either the active beverage (CGAs: 270 mg) or the placebo beverage group daily for 2 weeks. After a 2-week washout period, the participants consumed the other beverages. We assessed cognitive function at baseline and following the first treatment period using the Japanese version of CNS Vital Signs. CGAs significantly improved the scores for psychomotor speed, motor speed, and right and left finger tapping compared to placebo. In addition, processing speed scores improved significantly from baseline only after CGA intake. In conclusion, CGAs were confirmed to improve cognitive function over a short period of two weeks.

Effect of Short-Term Increase in Meal Frequency on Glucose Metabolism in Individuals with Normal Glucose Tolerance or Impaired Fasting Glucose: A Randomized Crossover Clinical Trial.

Effects of meal frequency on blood glucose levels and glucose metabolism were evaluated over 3 days in adult males with normal glucose tolerance (NGT, n = 9) or impaired fasting glucose (IFG, n = 9) in a randomized, crossover comparison study. Subjects were provided with an isocaloric diet 3 times daily (3M) or 9 times daily (9M). Blood glucose was monitored on Day 3 using a continuous glucose monitoring system, and subjects underwent a 75-g oral glucose tolerance test (OGTT) on Day 4. Daytime maximum blood glucose, glucose range, duration of glucose ≥180 mg/dL, and nighttime maximum glucose were significantly lower in the NGT/9M condition than in the NGT/3M condition. Similar findings were observed in the IFG subjects, with a lower daytime and nighttime maximum glucose and glucose range, and a significantly higher daytime minimum glucose in the 9M condition than in the 3M condition. The OGTT results did not differ significantly between NGT/3M and NGT/9M conditions. In contrast, the incremental area under the curve tended to be lower and the maximum plasma glucose concentration was significantly lower in the IFG/9M condition than in the IFG/3M condition. In IFG subjects, the 9M condition significantly improved glucose metabolism compared with the 3M condition. Higher meal frequency may increase glucagon-like peptide 1 secretion and improve insulin secretion.

Enteric peripheral neuroblastoma in a calf.

An 11-month-old female Japanese Black calf had showed chronic intestinal symptoms. A large mass surrounding the colon wall that was continuous with the colon submucosa was surgically removed. After recurrence and euthanasia, a large mass in the colon region and metastatic masses in the omentum, liver, and lung were revealed at necropsy. Pleomorphic small cells proliferated in the mass and muscular layer of the colon. The cells were positively stained with anti-doublecortin (DCX), PGP9.5, nestin, and neuron specific enolase (NSE). Thus, the diagnosis of peripheral neuroblastoma was made. This is the first report of enteric peripheral neuroblastoma in animals. Also, clear DCX staining signal suggested usefulness of DCX immunohistochemistry to differentiate the neuroblastoma from other small cell tumors in cattle.

Determination of the Absolute Configuration of a Monoglyceride Antibolting Compound and Isolation of Related Compounds from Radish Leaves (Raphanus sativus).

A monoglyceride (1) has been reported to possess an antibolting effect in radish (Raphanus sativus), but its absolute configuration at the C-2 position was not determined earlier. In this work, the absolute configuration of 1 was determined to be (2S), and it was also accompanied by one new (2) and two known monoglycerides (3 and 4). The chemical structure of 2 was determined as ß-(7'Z,10'Z,13'Z)-hexadecatrienoic acid monoglyceride (ß-16:3 monoglyceride). Qualitative and quantitative analytical methods for compounds 1-4 were developed, using two deuterium-labeled compounds (8 and 9) as internal standards. The results revealed a broader range of distribution of 1-4 in several annual winter crops. It was also found that these isolated compounds have an inhibitory effect on the root elongation of Arabidopsis thaliana seedlings at concentrations of 25 and 50 µM in the medium. However, the inhibitory effect of 1 was not dependent on coronatin-insensitive 1 (COI1) protein, which may suggest the involvement of an unidentified signaling system other than jasmonic acid signaling.

Effect of shortened sleep on energy expenditure, core body temperature, and appetite: a human randomised crossover trial.

The effects of sleep restriction on energy metabolism and appetite remain controversial. We examined the effects of shortened sleep duration on energy metabolism, core body temperature (CBT), and appetite profiles. Nine healthy men were evaluated in a randomised crossover study under two conditions: a 3.5-h sleep duration and a 7-h sleep duration for three consecutive nights followed by one 7-h recovery sleep night. The subjects' energy expenditure (EE), substrate utilisation, and CBT were continually measured for 48 h using a whole-room calorimeter. The subjects completed an appetite questionnaire every hour while in the calorimeter. Sleep restriction did not affect total EE or substrate utilisation. The 48-h mean CBT decreased significantly during the 3.5-h sleep condition compared with the 7-h sleep condition (7-h sleep, 36.75 ± 0.11 °C; 3.5-h sleep, 36.68 ± 0.14 °C; p = 0.016). After three consecutive nights of sleep restriction, fasting peptide YY levels and fullness were significantly decreased (p = 0.011), whereas hunger and prospective food consumption were significantly increased, compared to those under the 7-h sleep condition. Shortened sleep increased appetite by decreasing gastric hormone levels, but did not affect EE, suggesting that greater caloric intake during a shortened sleep cycle increases the risk of weight gain.

Lineage isolation in the face of active gene flow in the coastal plant wild radish is reinforced by differentiated vernalisation responses.

BACKGROUND: The respective role and relative importance of natural selection and gene flow in the process of population divergence has been a central theme in the speciation literature. A previous study presented conclusive evidence that wild radish on Japanese islands comprises two genetically isolated lineages: the southern and northern groups. However, a general understanding of the lineage isolation with frequent seed flow of the coastal plant species is still unclear. We surveyed nucleotide polymorphisms over 14 nuclear loci in 72 individuals across the Japan-Ryukyu Islands Arc to address the demographic history of wild radish utilising the isolation-with-migration (IM) model. In addition, we investigated the flowering times of individuals in different wild radish lineages, with and without cold exposure, to assess their respective vernalisation responses. RESULTS: Coalescent simulations suggested that divergence between the southern and northern lineages of wild radish began ~18,000 years ago, initially during the Last Glacial Maximum (LGM) period. The gene flow from the southern to northern groups was considerably higher than that in the opposite direction, indicating effective dispersal of viable seeds via the northward Kuroshio Current. Our greenhouse experiments indicated that cold exposure was not required for flowering in the southern group, but could advance the date of flowering, suggesting that vernalisation would be facultative in the southern group. In contrast, the northern group was either unable to flower or flowered later without prior cold exposure, and thus had an obligate requirement for cold treatment. CONCLUSIONS: The south-north lineage divergence in wild radish could be triggered by a directional change in the sea current during the ice age, despite gene flow due to the high dispersability and longevity of seeds. We also found that temperature profoundly affected the vernalisation responses of wild radish, which may repress reproductive success and ultimately drive and reinforce intra-specific differentiation between the two lineages of wild radish. This study provides new insights into the maintenance of lineage differentiation with on-going gene flow in coastal plants.

Distinct Phylogeographic Structures of Wild Radish (Raphanus sativus L. var. raphanistroides Makino) in Japan.

Coastal plants with simple linear distribution ranges along coastlines provide a suitable system for improving our understanding of patterns of intra-specific distributional history and genetic variation. Due to the combination of high seed longevity and high dispersibility of seeds via seawater, we hypothesized that wild radish would poorly represent phylogeographic structure at the local scale. On the other hand, we also hypothesized that wild radish populations might be geographically differentiated, as has been exhibited by their considerable phenotypic variations along the islands of Japan. We conducted nuclear DNA microsatellite loci and chloroplast DNA haplotype analyses for 486 samples and 144 samples, respectively, from 18 populations to investigate the phylogeographic structure of wild radish in Japan. Cluster analysis supported the existence of differential genetic structures between the Ryukyu Islands and mainland Japan populations. A significant strong pattern of isolation by distance and significant evidence of a recent bottleneck were detected. The chloroplast marker analysis resulted in the generation of eight haplotypes, of which two haplotypes (A and B) were broadly distributed in most wild radish populations. High levels of variation in microsatellite loci were identified, whereas cpDNA displayed low levels of genetic diversity within populations. Our results indicate that the Kuroshio Current would have contributed to the sculpting of the phylogeographic structure by shaping genetic gaps between isolated populations. In addition, the Tokara Strait would have created a geographic barrier between the Ryukyu Islands and mainland Japan. Finally, extant habitat disturbances (coastal erosion), migration patterns (linear expansion), and geographic characteristics (small islands and sea currents) have influenced the expansion and historical population dynamics of wild radish. Our study is the first to record the robust phylogeographic structure in wild radish between the Ryukyu Islands and mainland Japan, and might provide new insight into the genetic differentiation of coastal plants across islands.

The radish genome and comprehensive gene expression profile of tuberous root formation and development.

Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.

Demographic histories of adaptively diverged riparian and non-riparian species of Ainsliaea (Asteraceae) inferred from coalescent analyses using multiple nuclear loci.

BACKGROUND: Understanding demographic histories, such as divergence time, patterns of gene flow, and population size changes, in ecologically diverging lineages provide implications for the process and maintenance of population differentiation by ecological adaptation. This study addressed the demographic histories in two independently derived lineages of flood-resistant riparian plants and their non-riparian relatives [Ainsliaea linearis (riparian) and A. apiculata (non-riparian); A. oblonga (riparian) and A. macroclinidioides (non-riparian); Asteraceae] using an isolation-with-migration (IM) model based on variation at 10 nuclear DNA loci. RESULTS: The highest posterior probabilities of the divergence time parameters were estimated to be ca. 25,000 years ago for A. linearis and A. apiculata and ca. 9000 years ago for A. oblonga and A. macroclinidioides, although the confidence intervals of the parameters had broad ranges. The likelihood ratio tests detected evidence of historical gene flow between both riparian/non-riparian species pairs. The riparian populations showed lower levels of genetic diversity and a significant reduction in effective population sizes compared to the non-riparian populations and their ancestral populations. CONCLUSIONS: This study showed the recent origins of flood-resistant riparian plants, which are remarkable examples of plant ecological adaptation. The recent divergence and genetic signatures of historical gene flow among riparian/non-riparian species implied that they underwent morphological and ecological differentiation within short evolutionary timescales and have maintained their species boundaries in the face of gene flow. Comparative analyses of adaptive divergence in two sets of riparian/non-riparian lineages suggested that strong natural selection by flooding had frequently reduced the genetic diversity and size of riparian populations through genetic drift, possibly leading to fixation of adaptive traits in riparian populations. The two sets of riparian/non-riparian lineages showed contrasting patterns of gene flow and genetic differentiation, implying that each lineage showed different degrees of reproductive isolation and that they had experienced unique evolutionary and demographic histories in the process of adaptive divergence.

Isolation and characterization of microsatellite loci in the beach pea, Lathyrus japonicus (Fabaceae), in Japan.

PREMISE OF THE STUDY: Microsatellite loci were developed for Lathyrus japonicus (Fabaceae) to evaluate genetic diversity, population genetic structure, and demographic patterns of this species in Japan. METHODS AND RESULTS: In total, eight primer sets were identified in Japanese populations of L. japonicus and polymorphisms were evaluated in 83 individuals from three populations, including the northern and southern geographic range in Japan. The number of alleles per locus ranged from four to 20, and gene diversity per locus ranged from 0.636 to 0.935 among three populations. In addition, six of the eight loci could be successfully amplified in L. pratensis. CONCLUSIONS: These markers will be useful for studies of genetic variations, genetic structure, and gene flow of L. japonicus, which is important to investigate the demographic patterns of this coastal plant. In addition, the primers are useful for further genetic structure analysis of L. pratensis.

Ecological barriers to gene flow between riparian and forest species of Ainsliaea (Asteraceae).

Understanding the role of habitat-associated adaptation in reducing gene flow resulting in population differentiation and speciation is a major issue in evolutionary biology. We demonstrate a significant role for habitat divergence in species isolation between two naturally hybridizing riparian and nonriparian plants, Ainsliaea faurieana and A. apiculata (Asteraceae), on Yakushima Island, Japan. By analyzing the fine-scale population structure at six sympatric sites, we found that variations in leaf shape, geography, light conditions, and genotype were strongly correlated across riverbank-forest transitions. No evidence of effective gene flow was found between the two species across the majority of the transition zones, although the NewHybrid clustering analysis confirmed interspecific hybridization. However, a relatively high level of gene flow was observed across one zone with a more diffuse ecotone and intermediate flooding and light conditions, possibly generated by human disturbances. These results suggest that the barriers to gene flow between the riparian and forest species are primarily ecological. Additional common garden experiments indicated that the two species are adaptively differentiated to contrasting flooding and light environments. Overall, our study suggests that adaptations to different habitats can lead to the formation of reproductive isolating barriers and the maintenance of distinct species boundaries.

Molecular phylogeny and habitat diversification of the genus Farfugium (Asteraceae) based on nuclear rDNA and plastid DNA.

BACKGROUND AND AIMS: Farfugium (Asteraceae) is a small genus that contains the two species F. japonicum and F. hiberniflorum and is distributed along a long archipelago in east Asia. The common taxon, F. japonicum, includes three varieties associated with a wide range of habitats, including forest understorey (sciophytes), coastal crag (heliophytes) and riverbed (rheophytes). Leaf shape is an important taxonomic character within this genus and is associated with the habitat. METHODS: Twenty populations that included all Farfugium taxa were collected throughout its range. Leaf morphology was measured to determine differences amongst the taxa. Phylogenetic analyses based on sequences of the internal transcribed spacer of nuclear rDNA and four plastid DNA regions (matK, trnL-trnF, trnH-psbA and rpl20-rps12) were conducted separately. KEY RESULTS: Leaf morphology was significantly different amongst taxa, but morphological variations were partly explained by adaptation to certain environmental conditions that each population inhabited. Molecular phylogenies for the nDNA internal transcribed spacer and cpDNA were consistent in classifying F. hiberniflorum and the Taiwanese var. formosanum, whilst suggesting polyphyletic origins for the rheophyte, sciophyte and heliophyte taxa. All samples from the southern Ryukyus (Japan) and Taiwan clustered into a monophyletic group, which corroborates the land configuration theory involving Quaternary land-bridge formation and subsequent fragmentation into islands. The incongruence between the two DNA datasets may imply traces of introgressive hybridization and/or incomplete lineage sorting. CONCLUSIONS: The occurrence of rheophyte, sciophyte and heliophyte plants within Farfugium may be attributable to their isolation on islands and subsequent adaptation to the riparian, coastal crag and forest understorey environments, following their migration over the Quaternary land-bridge formation along their distribution range. Nearly identical DNA sequences coupled with highly divergent morphologies amongst these taxa suggest that diversification was rapid.

Multiple spatial scale patterns of genetic diversity in riparian populations of Ainsliaea faurieana (Asteraceae) on Yakushima Island, Japan.

Habitat and geographical features of river systems strongly influence gene flow and spatial genetic patterning in riparian plant populations. We investigated the patterns of genetic diversity within and among populations of Ainsliaea faurieana relative to different spatial conditions (along a river, among rivers, and among regions on an island), based on nuclear and chloroplast microsatellite DNA variations. Within an individual river system, we found higher haplotype diversities in downstream populations, and in a Bayesian analysis of recent migration, we detected unidirectional gene movements from upstream to downstream, indicating water-mediated dispersal along the river. Mantel tests detected no isolation-by-distance in genetic variation, suggesting the maintenance of a metapopulation with wide-range seed dispersal by water. Moreover, the observed high level of genetic differentiation, especially in the cpDNA (F(ST) = 0.539), indicated a metapopulation structure with frequent extinction and colonization. On a larger scale, we found high population differentiation and clear genetic structuring among regions, suggesting that gene flow was restricted by geographical features (mountains separating river systems) for relatively long periods. Our findings of genetic structures based on different spatial conditions elucidated patterns and ranges of historical and contemporary gene movement in a plant species that is persistent in extremely disturbed riparian environments.

Dural arteriovenous fistula in the anterior cranial fossa: four case reports.

Three of 4 cases of dural arteriovenous fistulas (DAVFs) in the anterior cranial fossa were detected incidentally by magnetic resonance (MR) imaging, and one case manifested as intracerebral hemorrhage. Cerebral angiography revealed fistulas located in the anterior cranial fossa. Three patients underwent surgery, and the fistulas were successfully obliterated. One patient with nonruptured DAVF requested conservative medical management. Incidental detection of asymptomatic or nonruptured DAVFs in the anterior cranial fossa has increased with the wider use of MR imaging. Increase in the size of a venous varix is the indicator for aggressive therapeutic intervention in a patient receiving conservative medical management for asymptomatic or nonruptured DAVFs in the anterior cranial fossa.

Phylogeny and biogeography of the genus Ainsliaea (Asteraceae) in the Sino-Japanese region based on nuclear rDNA and plastid DNA sequence data.

BACKGROUND AND AIMS: The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species. METHODS: Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers. KEY RESULTS: Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1.1 MYA during the early Pleistocene. CONCLUSIONS: The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies.

The blood pressure-lowering effect and safety of chlorogenic acid from green coffee bean extract in essential hypertension.

Chlorogenic acids (CGA) in green coffee bean extract (GCE) reduce blood pressure in spontaneously hypertensive rats and humans. The authors examined the blood pressure-lowering effect and safety of CGA in patients with mild hypertension through a placebo-controlled, randomized clinical trial. Subjects (n = 28) were randomized to receive treatment with CGA (140 mg/day) from GCE or placebo. Blood pressure, pulse rate, body mass index, routine blood test, hematochemistry, urinalysis, and subjective symptoms were recorded throughout the study. In the CGA group, but not the placebo group, blood pressure (systolic and diastolic) decreased significantly during the ingestion period. There was no difference in body mass index and pulse rate between groups, nor were there any apparent side effects. Thus, CGA from GCE is effective in decreasing blood pressure and safe for patients with mild hypertension.