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.

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.

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.

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.

Highly enantioselective catalytic 1,3-dipolar cycloadditions of α-alkyl diazoacetates: efficient synthesis of functionalized 2-pyrazolines.

Highly enantioselective 1,3-dipolar cycloaddition reactions of α-substituted diazoacetates are accomplished by catalysis of the chiral oxazaborolidinium ion. Functionalized 2-pyrazolines are synthesized in high to excellent enantiomeric ratios (up to >99 : 1). The synthetic utility of 2-pyrazoline was expanded via preparation of 2,4-diamino ester compounds bearing a chiral quaternary carbon center.

Catalytic enantioselective carbon insertion into the ß-vinyl C-H bond of cyclic enones.

Chiral oxazaborolidinium ion-catalyzed Csp(2)-H functionalization of enones using diazoacetate has been developed. Various ß-substituted cyclic enones were synthesized in high yield (up to 99%) with high to excellent enantioselectivity (up to 99% ee). The synthetic utility of this reaction was demonstrated by the formal synthesis of (+)-epijuvabione.

Isolation and characterization of novel Ty1-copia-like retrotransposons from lily.

Species of the genus Lilium are well known for their large genomes. Although expansion of noncoding repeated DNA is believed to account for this genome size, retroelement del Ty3-gypsy is the only one described so far in the genus Lilium. We isolated Ty1-copia elements from Lilium longiflorum and named them LIREs (lily retrotransposons). The long terminal repeats, primer binding site, and polypurine tract sequences are highly similar among the LIRE elements, indicating that they are in the same lineage. Although the protein-coding regions were highly decayed, the sequence motifs of the integrase, reverse transcriptase, and RNase H domains were identifiable as belonging to the order of Ty1-copia elements. Phylogenetic analysis and primer binding site sequences revealed that these elements belonged to the Ale lineage among the six lineages of plant Ty1-copia elements. Base substitutions in the long terminal repeats estimated that the integration times of the LIRE Ty1-copia elements were between 0.7 and 5.5 mya. In situ hybridization showed that the LIRE elements were present in all the chromosomes of L. longiflorum and L. lancifolium, but absent in centromeres, telomeres, and 45S rRNA sites in both species. The LIRE elements were present very abundantly in species of the genus Lilium, but absent in other genera of the family Liliaceae, implying that the LIRE elements might have contributed to the expansion of the genome in the genus Lilium.

Asymmetric synthesis of α-alkylidene-ß-hydroxy-γ-butyrolactones via enantioselective tandem Michael-aldol reaction.

A simple and efficient method for the asymmetric synthesis of α-alkylidene-ß-hydroxy-γ-butyrolactones and related natural products was developed on the basis of the catalytic asymmetric tandem Michael-aldol reaction and simple transformations. The synthetic utility of this method was illustrated by the facile synthesis of trisubstituted γ-butyrolactone natural products.

Total synthsis of (+)-ambuic acid: α-bromination with 1,2-dibromotetrachloroethane.

Total synthesis of (+)-ambuic acid has been accomplished from the readily available stereocontrolled Diels-Alder adduct of cyclopentadiene and iodo-1,4-benzoquinone monoketal through an efficient series of steps. A new method for the highly commendable synthesis of α-brominated Diels-Alder adduct is described.

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.

Fabrication of alumina-based metal nanocomposites by pressureless sintering and their mechanical properties.

The processing conditions to prepare nano-sized Cu and Mo dispersed Al2O3 (Al2O3/Cu and Al2O3/Mo) composites by pressureless sintering were explored. The composite powders of Al2O3/Cu and Al2O3/Mo were obtained by the hydrogen reduction of Al2O3/CuO and Al2O3/MoO3 powder mixtures and consolidated by pressureless sintering using infrared heating furnace with a heating rate of 200 degrees C/min. SEM and TEM analyses for the composite showed that the nano-sized metal particles were well distributed and situated on the grain boundaries of the Al2O3 matrix. The nanocomposites, sintered at 1300 to 1500 degrees C for 4 min, showed the relative density of above 90%. Maximum hardness of 16.1 GPa was obtained in Al2O3/Cu nanocomposites with sintering additive of 1 wt% MgO. The sintered nanocomposites exhibited the enhanced fracture toughness of above 4.5 MPa x m(1/2), compared with monolithic Al2O3. The mechanical properties were discussed in terms of observed microstructural characteristics.

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.

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress.

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0MHz ultrasound at 1.0W/cm2 for 60s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.

Construction of artificial promoters sensitively responsive to sonication in vitro.

PURPOSE: To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro. METHODS: The binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay. RESULTS: Of 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm(2) for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication. CONCLUSIONS: A promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

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.