Thymocyte Development of Humanized Mice Is Promoted by Interactions with Human-Derived Antigen Presenting Cells upon Immunization.

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.

CPUE standardization for southern bluefin tuna (Thunnus maccoyii) in the Korean tuna longline fishery, accounting for spatiotemporal variation in targeting through data exploration and clustering.

Accounting for spatial and temporal variation in targeting is a concern in many catch per unit effort (CPUE) standardization exercises. In this study we standardized southern bluefin tuna (Thunnus maccoyii, SBT) CPUE from the Korean tuna longline fishery (1996-2018) using generalized linear models (GLMs) with operational set by set data. Data were first explored to investigate the operational characteristics of Korean tuna longline vessels fishing for SBT, such as the spatial and temporal distributions of effort, and changes in the nominal catch rates among major species and species composition. Then we estimated SBT CPUE by area used for the stock assessment in the CCSBT (Commission for the Conservation of Southern Bluefin Tuna) and identified two separate areas in which Korean tuna longline vessels have targeted SBT and albacore tuna (T. alalunga), with targeting patterns varying spatially, seasonally and longer term. We applied two approaches, data selection and cluster analysis of species composition, and compared their ability to address concerns about the changing patterns of targeting through time. Explanatory variables for the GLM analyses were year, month, vessel identifier, fishing location (5° cell), number of hooks, moon phase, and cluster. GLM results for each area suggested that location, year, targeting, and month effects were the principal factors affecting the nominal CPUE. The standardized CPUEs for both areas decreased until the mid-2000s and have shown an increasing trend since that time.

Effects of different Bacillus licheniformis and Bacillus subtilis ratios on nutrient digestibility, fecal microflora, and gas emissions of growing pigs.

The objective of this study was to evaluate the effects of different mixing ratios of Bacillus licheniformis and Bacillus subtilis in diets on nutrient digestibility, fecal microflora, and odor gas emissions of growing pigs. A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows with average body weight (BW) of 41.2 ± 0.7 kg were randomly allotted four diets over four periods in a 4 × 4 Latin square design. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). Dietary probiotic supplementation showed higher crude protein (CP) digestibility values and lower Escherichia coli counts in fecal samples than the CON group (p < 0.05). There was no significant difference in NH3 or H2S emission until day 3. The positive effect of H2S and NH3 emissions was detected earlier with the L4S6 and L5S5 compared to the L6S4, which had a lower ratio of B. subtilis. Both the L4S6 and L5S5 probiotic complexes significantly decreased the fecal H2S and NH3 emission in days 4 and 6 (p < 0.05). On day 7, all probiotic complexes decreased (p < 0.05) H2S and NH3 emissions than the CON group. Our results agreed that the dietary supplementation of Bacillus licheniformis and Bacillus subtilis complexes in growing pigs can significantly improve CP digestibility and reduce fecal E. coli counts, NH3 and H2S emissions. Notably, the higher mixing ratio of Bacillus subtilis in probiotic supplementation is more effective in reducing the odor of manure.

Shallow population structure of southern bluefin tuna, Thunnus maccoyii (Castelnau, 1872) between Indian and Atlantic Oceans inferred from mitochondrial DNA sequences.

Southern bluefin tuna (Thunnus maccoyii Castelnau, 1872) is distributed across most of the southern temperate ocean and migrates extensively between 30°S and 50°S. Since T. maccoyii has been continually and heavily exploited, it is necessary to investigate the genetic diversity, population structure and demographic history of T. maccoyii for effective management and conservation. Thirty-seven gonad tissues of T. maccoyii were sampled from two locations, which were in the eastern Indian Ocean and the eastern Atlantic Ocean, by scientific observers onboard Korean T. maccoyii longline vessels in 2015. We compared 1240-bp sequences of combined mitochondrial DNA (mtDNA) from the cytochrome c oxidase subunit I (COI, 504-bp) and control region (CR, 736-bp) sequences. The pairwise fixation index (F ST) and maximum-likelihood tree showed that two clades (A and B) were formed regardless of locations. Clade A occurred more commonly than clade B in both localities: the occurrence ratio of clade A was 69% in the Indian Ocean, and 79% in the Atlantic Ocean, respectively. Our findings suggest that a historic differentiation event may have occurred in T. maccoyii, but recently the connectivity between the two oceans may be possible in T. maccoyii populations.

Epigenetic Variation Induced by Gamma Rays, DNA Methyltransferase Inhibitors, and Their Combination in Rice.

DNA methylation plays important roles in the regulation of gene expression and maintenance of genome stability in many organisms, including plants. In this study, we treated rice with gamma rays (GRs) and DNA methyltransferase inhibitors (DNMTis) to induce variations in DNA methylation and evaluated epigenetic diversity using methylation-sensitive amplified polymorphism (MSAP) and transposon methylation display (TMD) marker systems. Comparative and integrated analyses of the data revealed that both GRs and DNMTis alone have epimutagenic effects and that combined treatment enhanced these effects. Calculation of methylation rates based on band scoring suggested that both GRs and DNMTis induce epigenetic diversity by demethylation in a dose-dependent manner, and combined treatment can induce variations more synergistically. The difference in the changes in full and hemi-methylation rates between MSAP and TMD is presumed to be caused by the different genomic contexts of the loci amplified in the two marker systems. Principal coordinate, phylogenic, and population structure analyses commonly yielded two clusters of individuals divided by DNMTi treatment. The clustering pattern was more apparent in TMD, indicating that DNMTis have a stronger effect on hypermethylated repetitive regions. These findings provide a foundation for understanding epigenetic variations induced by GRs and DNMTis and for epigenetic mutation breeding.

Cytological variations and long terminal repeat (LTR) retrotransposon diversities among diploids and B-chromosome aneuploids in Lilium amabile Palibin.

BACKGROUND: B chromosomes are supernumerary chromosomes found in numerous plant species, including in the genus Lilium. Lilium amabile, an endemic Korean Lilium species, carries B chromosomes which are highly variable in terms of numbers and shape among the accessions collected throughout the Korea. Class 1 retrotransposons are highly abundant in the genome of Lilium species, but their biological functions are still obscure. Lilium species were known to hold high diversities derived from retrotransposons. OBJECTIVE: In this study, genetic diversities among the L. amabile accessions were analyzed to better understand relationships between genetic variations and cytological diversities. METHODS: Chromosomes were prepared from 95 L. amabile accessions for cytological identification. Genetic variations were analyzed by inter-retrotransposon amplified polymorphism (IRAP), and genetic differentiation was evaluated via Tajima's D neutrality and FST analyses. Population structure and phylogenetic analyses were also carried out. RESULTS: The L. amabile accessions were classified into 11 cytotypes by the chromosome constitutions. Genetic diversity measured by IRAP analysis revealed high genetic diversity among the accessions. In the joint analysis of cytological variation with genetical variation, IRAP diversity was not related to the cytological diversities of diploid and aneuploids among L. amabile accessions, and genetic differentiation was not obvious. Moreover, the geographical distribution of L. amabile was not related to either IRAP diversity or cytological diversity. CONCLUSION: The B chromosome-carrying aneuploids occurred randomly among diploids throughout Korea, and IRAP diversification predated L. amabile dispersion in Korea without genetic differentiation.

Induction of APOBEC3B cytidine deaminase in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL). Following viral infection with HTLV-1, certain infected cells exhibit clonal proliferation. Additional genetic and epigenetic changes in these clonally proliferating cells provide them with the selective advantage of growth, which eventually results in ATL. The precise mechanism, however, has yet to be completely elucidated. It has previously been established that APOBEC3 enzymes are potent host-antiviral restriction factors. Conversely, previous studies have reported that the A3B level is increased in tumor virus infections, such as those caused by HBV and HPV, suggesting that A3B exerts a function as a mutagen. Therefore, the present study analyzed the expression of APOBEC3 family members in various HTLV-1 infection states. No significant differences were observed in the expression between healthy donors and patients with HTLV-1-associated myelopathy. Although no significant changes in the expressions of A3C, A3D, A3F and A3G between uninfected and HTLV-1-infected mice were observed, an increased A3B expression was observed in a short-term humanized mouse model following HTLV-1 infection. In a long-term humanized mouse model following HTLV-1 infection, the gene expression array data exhibited an apparent increase in A3B and CADM1, which are indicators of ATL. Collectively, the results of the present study suggest that A3B is likely involved in the development of ATL in HTLV-1-infected humanized mice.

Characterization of chloroplast genomes of Alnus rubra and Betula cordifolia, and their use in phylogenetic analyses in Betulaceae.

OBJECTIVE: Betulaceae is a relatively small birch family that comprises about 160 deciduous trees and shrubs. Chloroplast (cp) genome sequencing of Alnus rubra and Betula cordifolia was carried out to elucidate their molecular features and phylogenetic relationship among species in Betulaceae family. METHODS: Chloroplast genome sequencing was carried out using next generation sequencing method. Molecular and genomic features of the two cp genomes were characterized with other cp genomes in Betulaceae. Also, molecular phylogenetic analysis was performed using the whole cp genome sequences. RESULTS: The average cp genome length was 160,136 bp among the Betulaceae species. Base compositions of the cp genomes were skewed toward a high AT ratio, with an average of 63.4%. We identified 117 different genes 83 with protein coding, 4 with ribosomal RNA, and 30 with tRNA. Eighteen genes contained introns which were conserved among the cp genomes of all Betulaceae. We mined 82 SSRs from the cp genomes of A. rubra, A. cordifolia, and A. nana. The SSRs were variable in motif repeat numbers and presence/absence among the cp genomes. CONCLUSION: Chloroplast genome-wide sequence comparison from 11 Betulaceae species and one cp genome of evergreen oak revealed that the patterns of sequence variations were congruent with two subfamily classification Betuloideae (Alnus and Betula) and Corylaceae (Corylus, Ostrya, and Carpinus). Subsequent phylogenetic analysis also supports the sub-classifications of these species.

Ty3/Gypsy retrotransposons in the Pacific abalone Haliotis discus hannai: characterization and use for species identification in the genus Haliotis.

Transposable elements are highly abundant elements that are present in all eukaryotic species. Here, we present a molecular description of abalone retrotransposon (Abret) elements. The genome of Haliotis discus hannai contains 130 Abret elements which were all Ty3/Gypsy retrotransposons. The Ty1/Copia elements were absent in the H. discus hannai genome. Most of the elements were not complete due to sequence truncation or coding region decay. However, three elements Abret-296, Abret-935, and Abret-3259 had most of the canonical features of LTR (long terminal repeat)-retrotransposons. There were several reading frame shifts in Abret-935 and Abret-3259 elements. Surprisingly, phylogenetic analysis indicated that all of the elements belonged to the Osvaldo lineage. The sequence divergence between LTRs revealed that the Abret elements were mostly active within 2 million years ago. Abret elements were used as molecular markers in SSAP analyses, which allowed clear distinction of different species in the genus Haliotis. The polymorphic markers were converted into SCAR markers for use in species identification by simple PCR in the Haliotis genus.

Retrotransposons in Betula nana, and interspecific relationships in the Betuloideae, based on inter-retrotransposon amplified polymorphism (IRAP) markers.

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined 'N' bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.

Evaluation of the secretion and release of vascular endothelial growth factor from two-dimensional culture and three-dimensional cell spheroids formed with stem cells and osteoprecursor cells.

BACKGROUND: Co-culture has been applied in cell therapy, including stem cells, and has been reported to give enhanced functionality. OBJECTIVES: In this study, stem-cell spheroids were formed in concave micromolds at different ratios of stem cells to osteoprecursor cells, and the amount of secretion of vascular endothelial growth factor (VEGF) was evaluated. MATERIAL AND METHODS: Gingiva-derived stem cells and osteoprecursor cells in the amount of 6 × 105 were seeded on a 24-well culture plate or concave micromolds. The ratios of stem cells to osteoprecursor cells included: 0:4 (group 1), 1:3 (group 2), 2:2 (group 3), 3:1 (group 4), and 4:0 (group 5). RESULTS: The morphology of cells in a 2-dimensional culture (groups 1-5) showed a fibroblast-like appearance. The secretion of VEGF increased with the increase in stem cells, and a statistically significant increase was noted in groups 3, 4 and 5 when compared with the media-only group (p < 0.05). Osteoprecursor cells formed spheroids in concave microwells, and no noticeable change in the morphology was noted with the increase in stem cells. Spheroids containing stem cells were positive for the stem-cell markers SSEA-4. The secretion of VEGF from cell spheroids increased with the increase in stem cells. CONCLUSIONS: This study showed that cell spheroids formed with stem cells and osteoprecursor cells with different ratios, using microwells, had paracrine effects on the stem cells. The secretion of VEGF increased with the increase in stem cells. This stem-cell spheroid may be applied for tissue-engineering purposes.

Development of CACTA transposon derived SCAR markers and their use in population structure analysis in Zea mays.

Molecular marker technologies have proven to be an important breakthrough for genetic studies, construction of linkage maps and population genetics analysis. Transposable elements (TEs) constitute major fractions of repetitive sequences in plants and offer a wide range of possible areas to be explored as molecular markers. Sequence characterized amplified region (SCAR) marker development provides us with a simple and time saving alternative approach for marker development. We employed the CACTA-TD to develop SCARs and then integrated them into linkage map and used them for population structure and genetic diversity analysis of corn inbred population. A total of 108 dominant SCAR markers were designed out of which, 32 were successfully integrated in to the linkage map of maize RIL population and the remaining were added to a physical map for references to check the distribution throughout all chromosomes. Moreover, 76 polymorphic SCARs were used for diversity analysis of corn accessions being used in Korean corn breeding program. The overall average polymorphic information content (PIC) was 0.34, expected heterozygosity was 0.324 and Shannon's information index was 0.491 with a percentage of polymorphism of 98.67%. Further analysis by associating with desirable traits may also provide some accurate trait specific tagged SCAR markers. TE linked SCARs can provide an added level of polymorphism as well as improved discriminating ability and therefore can be useful in further breeding programs to develop high yielding germplasm.

Enhanced Osteogenic Differentiation Potential of Stem-Cell Spheroids Created From a Coculture of Stem Cells and Endothelial Cells.

PURPOSE: This study was performed to fabricate stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells and to evaluate their viability and osteogenic differentiation potential. MATERIALS AND METHODS: Gingiva-derived stem cells were isolated, and stem cells and endothelial cells with a total of 6 × 10 cells were seeded into concave micromolds with different ratios of 6:0 (group 1), 4:2 (group 2), 3:3 (group 3), and 2:4 (group 4). RESULTS: Gingiva-derived stem cells and/or endothelia cells formed spheroids in concave microwells. There was a decreasing trend in the diameter of spheroids with increasing amounts of endothelial cells, but there were no statistically significant differences between the groups. The secretion of vascular endothelial growth factor from the spheroids was noted. The results of the alkaline phosphatase activity assays showed significantly higher values for groups 2, 3, and 4 when compared with the value of group 1. CONCLUSIONS: Conclusively, stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells using concave microwells enhanced osteogenic differentiation potential, and multicell spheroid-based cell delivery could be a simple and effective strategy for improving stem-cell therapy.

Chloroplast genomes of Lilium lancifolium, L. amabile, L. callosum, and L. philadelphicum: Molecular characterization and their use in phylogenetic analysis in the genus Lilium and other allied genera in the order Liliales.

Chloroplast (cp) genomes of Lilium amabile, L. callosum, L. lancifolium, and L. philadelphicum were fully sequenced. Using these four novel cp genome sequences and five other previously sequenced cp genomes, features of the cp genomes were characterized in detail among species in the genus Lilium and other related genera in the order Liliales. The lengths and nucleotide composition showed little variation. No structural variation was found among the cp genomes in Liliales. Gene contents were conserved among four newly sequenced cp genome in Lilium species, the only differences being in two pseudogenes. We identified 112 genes in 13 functional categories, 18 of which carried introns that were conserved among the species in Liliales. There were 16-21 SSR loci (>12 bp, >3 repeats) in the cp genomes in Lilium and the genomic locations of these loci were highly variable among the species. Average mutations were 15 SNPs per 1kb and 5 indels per 1kb, respectively, in the cp genomes of the newly sequenced four Lilium species. Phylogenetic classifications revealed some discrepancies between trees based on the cp genomes and previous classifications based on the morphology and geographic distributions.

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×105 (group A) or 8×105 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.

Evaluation of the shape, viability, stemness and osteogenic differentiation of cell spheroids formed from human gingiva-derived stem cells and osteoprecursor cells.

The present study was performed to create stem cell spheroids from human gingiva-derived stem cells and osteoprecursor cells and to evaluate the maintenance of the stemness, the viability and osteogenic differentiation of the cell spheroids. Gingiva-derived stem cells were isolated, and a total of 6×105 stem cells and osteoprecursor cells were seeded into concave micromolds at various ratios. Gingiva-derived stem cells and/or osteoprecursor cells formed spheroids in concave microwells. The spheroids demonstrated a smaller diameter when the number of osteoprecursor cells seeded was lower. The majority of cells in the spheroids were identified to be live cells and the cell spheroids preserved viability throughout the experimental period. The cell spheroids, which contained stem cells, were positive for stem-cell markers. Cell spheroids in concave microwells demonstrated a statistically significant increase in alkaline phosphatase activity as time progressed (P<0.05). A statistically significant difference in phosphatase activity was observed in the stem cell alone group when compared with the osteoprecursor cell group at day 5 (P<0.05). Mineralized extracellular deposits were observed in each group after Alizarin Red S staining. Within the limits of the present study, cell spheroids from gingival cells and osteoprecursor cells maintained shape, viability, stemness and osteogenic differentiation potential.

Ribosomal DNA locus variation and REMAP analysis of the diploid and triploid complexes of Lilium lancifolium.

Lilium lancifolium Thunb. (2n = 2x = 24) is a cytologically conspicuous species with both diploids and triploids in nature. Cytological and molecular genetic analyses were carried out in both diploids and triploids that were collected from 55 geographical locations in Korea, Japan, and China. While the 5S rRNA gene loci were located at duplicated loci on the long arm of chromosome 2, the 45S rRNA gene loci were present in chromosomes 1, 2, 4, 6, 7, and 11. While the loci on chromosomes 1 and 7 were constant, the loci on chromosomes 2, 4, 6, 7, and 11 were variable in some plants so that the L. lancifolium accessions were grouped into 7 cytotypes in diploids and 12 cytotypes in triploids. REMAP marker analysis revealed that the diploids were classified into seven clusters, and the triploids were classified into a large cluster. Geographic, cytological, and genetic differentiations were not related in both the diploid and triploid accessions of L. lancifolium. Thus, current genetic variations occurred prior to the geographic differentiation in both diploids and triploids, and the 45S rDNA cytotype variations occurred after geographic differentiation in the current habitats of L. lancifolium.

Formation of size-controllable spheroids using gingiva-derived stem cells and concave microwells: Morphology and viability tests.

Human mesenchymal stem cells have previously been isolated and characterized from the gingiva, and gingiva-derived stem cells have been applied for tissue engineering purposes. The present study was performed to generate size-controllable stem cell spheroids using concave microwells. Gingiva-derived stem cells were isolated, and the stem cells of 1×105 (group A) or 2×105 (group B) cells were seeded in polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres was viewed under an inverted microscope, and the changes in the diameter and cell viability were analyzed. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A compared to group B. No significant changes in shape or diameter were noted with increases in incubation time. Cell viability was higher in group B at each time point when compared with group A. Within the limits of the study, the size-controllable stem cell spheroids could be generated from gingival cells using microwells. The shape of the spheroids and their viability were clearly maintained during the experimental periods.

LTR-retrotransposons and inter-retrotransposon amplified polymorphism (IRAP) analysis in Lilium species.

LTR-retrotransposons are ubiquitous and highly abundant in plant genomes. Moreover, LTR-retrotransposons can often cause genome obesity in plants. Although Lilium species have been known carrying large genomes among flowering plants, reports on the LTR-retrotransposons in Lilium species are rather limited. We isolated a novel Ty3/gypsy-like retrotransposon, LIRE-del, and two Ty1/copia-like retrotransposons, a LIRE-del and an unclassified, from a fosmid clone of Lilium longiflorum. Decayed internal ORF sequences indicated that they were non-autonomous elements. IRAP protocol was developed based on the LTR sequences of the isolated LTR-retrotransposons. Fourteen primer combinations showed clear distinctive PCR amplification bands that were highly informative in the analysis of species relationship among Lilium species. The phylogenetic relationship based on the IRAP profile revealed some discordant with phylogenetic studies based on the ITS sequences of 45S ribosomal gene and matK gene variations in a few species. Thus, the phylogenetic relationship among Lilium species may need to be re-evaluated with other tools such as cross compatibility and selectively neutral genetic markers.