Proteome Analysis of Human Natural Killer Cell Derived Extracellular Vesicles for Identification of Anticancer Effectors.

Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, ß-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.

Cytotoxic effects of ex vivo-expanded natural killer cell-enriched lymphocytes (MYJ1633) against liver cancer.

BACKGROUND: Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a targeted method of controlling the immune system against cancer. Despite their significant therapeutic potential, efficient methods to generate adequate numbers of NK cells are lacking and ex vivo-expansion and activation of NK cells is currently under intensive investigation. The primary purpose of this study was to develop an effective method for expansion and activation of the effector cells with high proportion of NK cells and increasing cytotoxicity against liver cancer in a short time period. METHODS: Expanded NK cell-enriched lymphocytes (NKL) designated as "MYJ1633" were prepared by using autologous human plasma, cytokines (IL-2, IL-12 and IL-18) and agonistic antibodies (CD16, CD56 and NKp46) without an NK cell-sorting step. The characteristics of NKL were compared to those of freshly isolated PBMCs. In addition, the cytotoxic effect of the NKL on liver cancer cell was examined in vitro and in vivo. RESULTS: The total cell number after ex vivo-expansion increased about 140-fold compared to that of freshly isolated PBMC within 2 weeks. Approximately 78% of the expanded and activated NKL using the house-developed protocol was NK cell and NKT cells even without a NK cell-sorting step. In addition, the expanded and activated NKL demonstrated potent cytotoxicity against liver cancer in vitro and in vivo. CONCLUSION: The house-developed method can be a new and effective strategy to prepare clinically applicable NKL for autologous NK cell-based anti-tumor immunotherapy.

Factors influencing COVID-19 vaccination intention among parents of children aged 5-11 years in South Korea: a cross-sectional study.

PURPOSE: The purpose of this study was to identify factors affecting parents' intentions to have their children aged 5-11 years vaccinated against coronavirus disease 2019 (COVID-19). METHODS: The participants of the study were 298 parents with children aged 5-11 years in South Korea. Data collection took place from October 20 to October 26, 2022 and used an online survey (Google Forms). Data were analyzed using descriptive statistics, the t test, analysis of variance, the Scheffé test, Pearson correlation coefficients, and hierarchical regression in IBM SPSS version 26.0. RESULTS: The factors influencing participants' vaccination intentions for their children aged 5-11 years were cognitive behavioral control (ß=.40, p<.001), attitudes (ß=.37, p<.001), subjective norms (ß=.20, p<.001), and awareness of whether their child could receive the COVID-19 vaccine (ß=.07, p=.016). The explanatory power of the regression equation was 89%. CONCLUSION: Parents' intentions to vaccinate their children against COVID-19 are influenced by their attitudes, subjective norms, and perceived behavioral control toward vaccines. Since parents are concerned about vaccine side effects, it is important to establish a trusted line of communication to keep them informed about vaccinations.

Correction: Genetic and chemical markers for authentication of three Artemisia species: A. capillaris, A. gmelinii, and A. fukudo.

[This corrects the article DOI: 10.1371/journal.pone.0264576.].

High-throughput discovery of plastid genes causing albino phenotypes in ornamental chimeric plants.

Chimeric plants composed of green and albino tissues have great ornamental value. To unveil the functional genes responsible for albino phenotypes in chimeric plants, we inspected the complete plastid genomes (plastomes) in green and albino leaf tissues from 23 ornamental chimeric plants belonging to 20 species, including monocots, dicots, and gymnosperms. In nine chimeric plants, plastomes were identical between green and albino tissues. Meanwhile, another 14 chimeric plants were heteroplasmic, showing a mutation between green and albino tissues. We identified 14 different point mutations in eight functional plastid genes related to plastid-encoded RNA polymerase (rpo) or photosystems which caused albinism in the chimeric plants. Among them, 12 were deleterious mutations in the target genes, in which early termination appeared due to small deletion-mediated frameshift or single nucleotide substitution. Another was single nucleotide substitution in an intron of the ycf3 and the other was a missense mutation in coding region of the rpoC2 gene. We inspected chlorophyll structure, protein functional model of the rpoC2, and expression levels of the related genes in green and albino tissues of Reynoutria japonica. A single amino acid change, histidine-to-proline substitution, in the rpoC2 protein may destabilize the peripheral helix of plastid-encoded RNA polymerase, impairing the biosynthesis of the photosynthesis system in the albino tissue of R. japonica chimera plant.

Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution.

Euonymus hamiltonianus and its relatives (Celastraceae family) are used for ornamental and medicinal purposes. However, species identification in Euonymus is difficult due to their morphological diversity. Using plastid genome (plastome) data, we attempt to reveal phylogenetic relationship among Euonymus species and develop useful markers for molecular identification. We assembled the plastome and nuclear ribosomal DNA (nrDNA) sequences from five Euonymus lines collected from South Korea: three Euonymus hamiltonianus accessions, E. europaeus, and E. japonicus. We conducted an in-depth comparative analysis using ten plastomes, including other publicly available plastome data for this genus. The genome structures, gene contents, and gene orders were similar in all Euonymus plastomes in this study. Analysis of nucleotide diversity revealed six divergence hotspots in their plastomes. We identified 339 single nucleotide polymorphisms and 293 insertion or deletions among the four E. hamiltonianus plastomes, pointing to abundant diversity even within the same species. Among 77 commonly shared genes, 9 and 33 were identified as conserved genes in the genus Euonymus and E. hamiltonianus, respectively. Phylogenetic analysis based on plastome and nrDNA sequences revealed the overall consensus and relationships between plastomes and nrDNAs. Finally, we developed six barcoding markers and successfully applied them to 31 E. hamiltonianus lines collected from South Korea. Our findings provide the molecular basis for the classification and molecular taxonomic criteria for the genus Euonymus (at least in Korea), which should aid in more objective classification within this genus. Moreover, the newly developed markers will be useful for understanding the species delimitation of E. hamiltonianus and closely related species.

Genetic and chemical markers for authentication of three Artemisia species: A. capillaris, A. gmelinii, and A. fukudo.

The genus Artemisia is an important source of medicines in both traditional and modern pharmaceutics, particularly in East Asia. Despite the great benefits of herbal medicine, quality assessment methods for these medicinal herbs are lacking. The young leaves from Artemisia species are generally used, and most of the species have similar morphology, which often leads to adulteration and misuse. This study assembled five complete chloroplast genomes of three Artemisia species, two accessions of A. gmelinii and A. capillaris, and one A. fukudo. Through comparative analysis, we revealed genomic variations and phylogenetic relationships between these species and developed seven InDel-based barcode markers which discriminated the tested species from each other. Additionally, we analyzed specialized metabolites from the species using LC-MS and suggested chemical markers for the identification and authentication of these herbs. We expect that this integrated and complementary authentication method would aid in reducing the misuse of Artemisia species.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

Diversity and authentication of Rubus accessions revealed by complete plastid genome and rDNA sequences.

Complete plastid genome (plastome) and ribosomal DNA (rDNA) sequences of three Rubus accessions (two Rubus longisepalus and one R. hirsutus) were newly assembled using Illumina whole-genome sequences. Rubus longisepalus Nakai and R. longisepalus var. tozawai, described as different varieties, have identical plastomes and rDNA sequences. The plastomes are 155,957 bp and 156,005 bp and the 45S rDNA transcription unit sizes are 5809 bp and 5811 bp in R. longisepalus and R. hirsutus, respectively. The 5S rDNA transcription unit is an identical 121 bp in three Rubus accessions. We developed three DNA markers to authenticate R. longisepalus and R. hirsutus based on plastome diversity. Phylogenomic analysis revealed that the Rubus species classified as two clades and R. longisepalus, R. hirsutus, and R. chingii are the most closely related species in clade 1.

Assessing the genetic and chemical diversity of Taraxacum species in the Korean Peninsula.

The genetic relationship between Taraxacum species, also known as the dandelion, is complicated because of asexual and mixed sexual apomictic reproduction. The usage of Taraxacum species in traditional medicines make their specialized metabolism important, but interspecific chemical difference has rarely been reported for the genus. In this study, we assembled the chloroplast genome and 45S rDNA of six Taraxacum species that occur in Korea (T. campylodes, T. coreanum, T. erythrospermum, T. mongolicum, T. platycarpum, and T. ussuriense), and performed a comparative analysis, which revealed their phylogenetic relationships and possible natural hybridity. We also performed a liquid chromatography-mass spectrometry-based phytochemical analysis to reveal interspecific chemical diversity. The comparative metabolomics analysis revealed that Taraxacum species could be separated into three chemotypes according to their major defensive specialized metabolites, which were the sesquiterpene lactones, the phenolic inositols, and chlorogenic acid derivatives. The CP DNA- and 45S rDNA-based phylogenetic trees showed a tangled relationship, which supports the notion of ongoing hybridization of wild Taraxacum species. The untargeted LC-MS analysis revealed that each Taraxacum plant exhibits species-specific defensive specialized metabolism. Moreover, 45S rDNA-based phylogenetic tree correlated with the hierarchical cluster relied on metabolite compositions. Given the coincidence between these analyses, we represented that 45S rDNA could well reflect overall nuclear genome variation in Taraxacum species.

The complete chloroplast genome sequence of Hosta capitata (Koidz.) Nakai (Asparagaceae).

Hosta capitata (Koidz.) Nakai is an herbaceous perennial plant belonging to the Asparagaceae family and has become a popular ornamental plant. In this study, the chloroplast genome sequence of H. capitata was completed by de novo assembly with whole genome sequence data. The chloroplast genome of H. capitata is 156,416 bp in length, which is composed of a large single-copy (LSC) of 84,788 bp, a small single-copy (SSC) of 18,206 bp, and a pair of inverted repeat regions (IRa and IRb) of 26,711 bp, as four distinct parts. In total, 114 genes were identified including 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis revealed that H. capitata has a close relationship with other Hosta species, H. minor and H. ventricosa, but is farther than the distance between them.

The complete chloroplast genome of Codonopsis lanceolata (Campanulaceae).

The complete chloroplast genome sequence of Codonopsis lanceolata was determined by next generation sequencing. The total length of chloroplast genome of C. lanceolata was 169,447 bp long, including a large single-copy (LSC) region of 85,253 bp, a small single-copy (SSC) region of 8060 bp, and a pair of identical inverted repeat regions (IRs) of 38,067 bp. A total of 110 genes was annotated, resulting in 79 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. The phylogenetic analysis of C. lanceolata with related chloroplast genome sequences in this study provided the taxonomical relationship of C. lanceolata in the genus Campanula.

The complete chloroplast genome of Caltha Palustris (Ranunculaceae).

The complete chloroplast genome sequence of Caltha palustris, a species of the Ranunculaceae family, was characterized from the de novo assembly of HiSeq (Illumina Co.) paired-end sequencing data. The chloroplast genome of C. palustris was 155,292 bp in length, with a large single-copy (LSC) region of 84,120 bp, a small single-copy (SSC) region of 18,342 bp, and a pair of identical inverted repeat regions (IRs) of 26,415 bp. The genome contained a total of 114 genes, including 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The phylogenetic analysis of C. palustris with 14 related species revealed the closest taxonomical relationship with Hydrastis canadensis in the Ranunculaceae family.

The complete chloroplast genome sequence of Magic Lily (Lycoris squamigera).

Magic Lily (Lycoris squamigera), belonging to the Amaryllidaceae family, is cultivated for ornamental and medicinal purposes. To characterize its genomic information, we obtained the complete chloroplast genome sequence of L. squamigera by assembling Illumina whole genome sequence data. The complete chloroplast genome is 158,482 bp in length which is composed of four unique regions, a large single copy region (LSC) of 86,454 bp, a small single copy region (SSC) of 18,500 bp, and a pair of inverted repeats (IR) of 26,764 bp. The genome annotation predicted 159 genes including 105 protein-coding genes, 46 tRNA genes, and 8 rRNA genes. Phylogenetic tree analysis revealed that L. squamigera clustered with Allium species belonging to the Amaryllidaceae family.

The complete chloroplast genome sequence of Ligularia fischeri (Ledeb.) Turcz. (Asteraceae).

We characterized the complete chloroplast genome sequence of Ligularia fischeri, a collection from Halla Mountain in Jeju Island, Korea. The plants are utilized as edible functional plant species harbouring useful antioxidant compounds in family Asteraceae. De novo assembly with whole genome sequencing data of L. fischeri completed the chloroplast genome of 151 133 bp long, which included two inverted repeats (IRs) blocks of 24 831 bp, separated by the large single-copy block of 83 238 bp and small single-copy block of 18 233 bp. The genome encoded 113 genes consisting of 80 protein-coding genes, 29 tRNA genes and 4 rRNA genes. Phylogenetic analysis with protein coding gene sequences of reported Asteraceae chloroplast genomes revealed a close relationship of L. fischeri with Jacobaea vulgaris, a weed species world-widely distributed.

The complete chloroplast genome sequence of a Korean indigenous ornamental plant Hydrangea serrata for. fertilis Nakai (Hydrangeaceae).

De novo assembly with whole genome sequencing data of Hydrangea serrata for. fertilis, a great ornamental landscape plant species worldwide, facilitated to generate the complete chloroplast genome sequence in this study. The complete sequence was a circular DNA molecule of 157 730 bp in length, containing the large single-copy (LSC) region of 86 789 bp, small single-copy (SSC) region of 18 711 bp and two inverted repeats (IRs) regions of 26 115 bp. The genome encoded 114 genes consisting of 80 protein-coding genes, 30 tRNA genes and four rRNA genes. Phylogenetic analysis with matK gene-coding sequences of 19 species in family Hydrangeaceae showed a close relationship of H. serrata for. fertilis Nakai with H. macrophylla.

The complete chloroplast genome sequence of the Taraxacum officinale F.H.Wigg (Asteraceae).

Taraxacum officinale is a distributed weedy plant used as a traditional medicinal herb belonging to the family Asteraceae. The complete chloroplast genome of T. officinale was generated by de novo assembly with whole genome sequence data. The chloroplast genome was 151 324 bp in length, which consisted of a large single copy region of 83 895 bp and a short single copy region of 18 549 bp separated by a pair of inverted repeat regions of 24 440 bp. The chloroplast genome contained 79 protein-coding genes, 29 tRNA genes and four rRNA genes. Phylogenetic analysis revealed that T. officinale was closely related to Lactuca sativa.

The complete chloroplast genome sequences of Artemisia gmelinii andArtemisia capillaris (Asteraceae).

In this study, complete chloroplast sequences of Artemisia gmelinii and Artemisia capillaris (the Asteraceae family), which have been used as herbal medicine in Korea, were characterized by de novo assembly with whole-genome sequence data. The genomes of A. gmelinii and A. capillaris were 151,318 bp and 151,056 bp in length, respectively. Both genomes harbored identical number of annotated genes, such as 80 protein coding genes, 4 rRNA genes and 30 tRNA genes. Phylogenetic tree revealed that both A. gmelinii and A. capillaris were closely grouped with other Artemisia species.

The complete chloroplast genome of Eclipta prostrata L. (Asteraceae).

Eclipta prostrata is an herbal medicinal plant belonging to the Asteraceae family. In this study, complete chloroplast genome sequence of the E. prostrata was characterized by de novo assembly using whole genome sequence data. The genome of E. prostrata was 151,757 bp in length, which was composed of large single copy region of 83,285 bp, small single copy region of 18,346 bp and a pair of inverted repeat regions of 25,063 bp. The genome harboured 80 protein coding sequences, 30 tRNA genes and 4 rRNA genes. We confirmed close taxonomic relationship between E. prostrata and Helianthus annuus through phylogenetic analysis with chloroplast protein-coding genes.

Complete chloroplast genome sequence of Artemisia fukudo Makino (Asteraceae).

In this study, a complete chloroplast genome sequence of Artemisia fukudo (Asteraceae family) was characterized by de novo assembly using whole genome sequence data. The chloroplast genome was 151,011 bp in length, comprising a large single-copy region of 82,751 bp, a small single copy region of 18,348 bp and a pair of inverted repeats of 24,956 bp. The genome contained 80 protein-coding genes, 4 rRNA genes and 30 tRNA genes. Phylogenetic tree revealed that A. fukudo was closely located in other Artemisia species, Artemisia montana and Artemisia frigida.