Comparative transcriptome analysis of the invasive weed Mikania micrantha with its native congeners provides insights into genetic basis underlying successful invasion.

BACKGROUND: Mikania micrantha H.B.K. (Asteraceae) is one of the world's most invasive weeds which has been rapidly expanding in tropical Asia, including China, while its close relative M. cordata, the only Mikania species native to China, shows no harm to the local ecosystems. These two species are very similar in morphology but differ remarkably in several ecological and physiological traits, representing an ideal system for comparative analysis to investigate the genetic basis underlying invasion success. In this study, we performed RNA-sequencing on the invader M. micrantha and its native congener M. cordata in China, to unravel the genetic basis underlying the strong invasiveness of M. micrantha. For a more robust comparison, another non-invasive congener M. cordifolia was also sequenced and compared. RESULTS: A total of 52,179, 55,835, and 52,983 unigenes were obtained for M. micrantha, M. cordata, and M. cordifolia, respectively. Phylogenetic analyses and divergence time dating revealed a relatively recent split between M. micrantha and M. cordata, i.e., approximately 4.81 million years ago (MYA), after their divergence with M. cordifolia (8.70 MYA). Gene ontology classifications, pathway assignments and differential expression analysis revealed higher representation or significant up-regulation of genes associated with photosynthesis, energy metabolism, protein modification and stress response in M. micrantha than in M. cordata or M. cordifolia. Analysis of accelerated evolution and positive selection also suggested the importance of these related genes and processes to the adaptability and invasiveness of M. micrantha. Particularly, most (77 out of 112, i.e. 68.75%) positively selected genes found in M. micrantha could be classified into four groups, i.e., energy acquisition and utilization (10 genes), growth and reproduction (13 genes), protection and repair (34 genes), and signal transduction and expression regulation (20 genes), which may have contributed to the high adaptability of M. micrantha to various new environments and the capability to occupy a wider niche, reflected in its high invasiveness. CONCLUSIONS: We characterized the transcriptomes of the invasive species M. micrantha and its non-invasive congeners, M. cordata and M. cordifolia. A comparison of their transcriptomes provided insights into the genetic basis of the high invasiveness of M. micrantha.

Overexpressing Bcl-2 enhances murine chimeric antigen receptor T cell therapy against solid tumor.

Chimeric antigen receptor T (CART) cell therapy has demonstrated promising potential in the treatment of hematologic malignancies. However, its application to solid tumors is limited due to the restrictive nature of the tumor microenvironment, resulting in functional failure and poor persistence of CART cells. Overexpression of Bcl-2 in human CART cells (hCART) has been found to significantly enhance their anti-apoptotic effects both in vitro and in vivo. Nevertheless, the evaluation of hCART cells in preclinical studies has predominantly relied on immunodeficient mice xenograft tumor models, making it challenging to assess the impact of hCART cells on normal tissues and the immune system. We established a murine CART (mCART) that overexpresses Bcl-2 and targets the epidermal growth factor receptor variant III (EGFRvIII), named EGFRvIII·mCART-Bcl2. It demonstrated superior proliferation, cytotoxicity, and anti-apoptotic capabilities in vitro. In an immunocompetent mouse model of abdominal metastasis of colorectal cancer, EGFRvIII·mCART-Bcl2 exhibited improved survival of CART in the abdomen, increased tumor clearance, and significantly prolonged overall mouse survival. In summary, our study provides evidence that the introduction of Bcl-2 into mCART cells can enhance their therapeutic efficacy against solid tumors while ensuring safety.

Population genetics of Camellia granthamiana, an endangered plant species with extremely small populations in China.

Introduction: Camellia, the largest genus of Theaceae, is well-known for having high economic values. Camellia granthamiana demonstrates large beautiful flowers with some primitive characters, such as multiple large and persistent bracteoles and sepals, was listed as Vulnerable species on the IUCN Red List. Methods: In this study, we investigated all possible records of the species, and sampled four natural populations and five cultivated individuals. By applying shallow-genome sequencing for nine individuals and RAD-seq sequencing for all the sampled 77 individuals, we investigated population genetic diversity and population structure of the species. Results and discussion: The results showed that the population sampled from Fengkai, previously identified as C. albogigias, possessed different plastid genome from other species possibly due to plastid capture; the species possesses strong population structure possibly due to the effect of isolation by distance, habitat fragmentation, and self-crossing tendency of the species, whose effective population size declined quickly in the past 4,000 years. Nevertheless, C. granthamiana maintains a medium level of genetic diversity within population, and significant differentiation was observed among the four investigated populations, it is anticipated that more populations are expected to be found and all these extant populations should be taken into instant protection.

Application of Elastin-like Polypeptide in Tumor Therapy.

Elastin-like polypeptides (ELPs) are stimulus-responsive artificially designed proteins synthesized from the core amino acid sequence of human tropoelastin. ELPs have good biocompatibility and biodegradability and do not systemically induce adverse immune responses, making them a suitable module for drug delivery. Design strategies can equip ELPs with the ability to respond to changes in temperature and pH or the capacity to self-assemble into nanoparticles. These unique tunable biophysicochemical properties make ELPs among the most widely studied biopolymers employed in protein purification, drug delivery, tissue engineering and even in tumor therapy. As a module for drug delivery and as a carrier to target tumor cells, the combination of ELPs with therapeutic drugs, antibodies and photo-oxidation molecules has been shown to result in improved pharmacokinetic properties (prolonged half-life, drug targeting, cell penetration and controlled release) while restricting the cytotoxicity of the drug to a confined infected site. In this review, we summarize the latest developments in the application methods of ELP employed in tumor therapy, with a focus on its conjugation with peptide drugs, antibodies and photosensitizers.

The complete chloroplast genome sequence of Camellia ptilophylla (Theaceae): a natural caffeine-free tea plant endemic to China.

Camellia ptilophylla is a natural caffeine-free tea plant endemic to China with high commercial and therapeutic values. Here, we report the complete chloroplast genome assembled using Illumina pair-end sequencing data. The chloroplast genome was 157,097 bp in length, with a large single copy (LSC) region of 86,631 bp, a small single copy (SSC) region of 18,286 bp, separated by two inverted repeat (IR) regions of 26,090 bp each. It contains a total of 132 genes, with an overall GC content of 37.3%. The phylogenetic analysis showed that C. ptilophylla is sister to a congeneric species, C. reticulata.

Characterization of the complete chloroplast genome of Camellia granthamiana (Theaceae), a Vulnerable species endemic to China.

Camellia granthamiana is a wild camellia resource endemic to China and is listed as a Vulnerable species globally. Here, we reported and characterized its complete chloroplast (cp) genome by using Illumina pair-end sequencing data. The total chloroplast genome size was 157,001 bp, including inverted repeats (IRs) of 26,042 bp, separated by a large single copy (LSC) and a small single copy (SSC) of 86,622 and 18,295 bp, respectively. A total of 131 genes, including 36 tRNA, 8 Rrna, and 87 protein-coding genes were identified. Phylogenetic analysis showed that C. granthamiana is sister to C. sinensis with 100% value support.

Development of polymorphic EST-SSR markers in Itea chinensis (Iteaceae) and cross-amplification in related species.

PREMISE OF THE STUDY: We isolated and characterized 16 expressed sequence tag-simple sequence repeat (EST-SSR) markers in Itea chinensis (Iteaceae), a common evergreen broadleaf tree, for future studies on the genetic diversity and spatial genetic structure of the species. METHODS AND RESULTS: Based on transcriptome data of I. chinensis, a total of 36 primer pairs were initially designed and tested. Of these, 16 were successfully amplified and showed clear polymorphism. For these markers, the number of alleles per locus varied from two to 15. The observed and expected heterozygosity ranged from 0 to 0.600 and 0.072 to 0.554, respectively. Furthermore, all loci were successfully cross-amplified in two congeneric species, I. oblonga and I. yangchunensis. CONCLUSIONS: The EST-SSR markers described here can be used to study the genetic diversity and phylogeographic patterns of I. chinensis and other related species in Itea.

Mummified Oligocene fruits of Schima (Theaceae) and their systematic and biogeographic implications.

The genus Schima includes about 20 species and is distributed only in southern China and adjacent areas of Asia. The previous molecular phylogenetic analysis suggested Schima is in the tribe Gordoniae, along with Gordonia and Franklinia. However, because few fossils have been reported, the biogeographic origin of Schima is still poorly known. In this paper mummified fossil fruits of Schima are described from the upper Oligocene Yongning Formation of the Nanning Basin, Guangxi, South China. In gross morphology, the new fossil species, Schima kwangsiensis, is similar to the extant S. superba by its pentacarpellate, loculicidally dehiscent capsules, 5 imbricate sepals, pedicels with bracteoles and marginally winged seeds. Due to its excellent preservation, the new species may provide sufficient details for understanding the early evolutionary and phytogeographic history of the genus. Morphological clustering analysis shows that the new fossil species is closely related to two extant species (S. wallichii and S. superba) in the genus, implying that they may belong to an ancient taxon that occurs earlier than the others. More importantly, this discovery represents the earliest record of this genus in Asia and it explicitly moves the fossil record back to the late Oligocene in this region.

Development of microsatellite markers for Eurya acuminatissima (Theaceae).

PREMISE OF THE STUDY: Sixteen microsatellite markers were developed to study the fine-scale spatial genetic structure of Eurya acuminatissima, a dioecious tree species of Theaceae endemic to southern China. METHODS AND RESULTS: A total of 30 primer pairs were initially designed and tested on the basis of the transcriptome data of E. acuminatissima, of which 16 were successfully amplified and showed clear polymorphism. For these microsatellites, one to 17 alleles per locus were identified. The observed and expected heterozygosities ranged from 0 to 1.000 and 0 to 0.903, respectively. CONCLUSIONS: The microsatellite markers described here can be used to study genetic diversity and spatial genetic structure of E. acuminatissima. Furthermore, all loci were successfully cross-amplified in a related species, E. auriformis.

Comparative Analysis of Transcriptomes in Rhizophoraceae Provides Insights into the Origin and Adaptive Evolution of Mangrove Plants in Intertidal Environments.

Mangroves are woody plants that grow at the interface between land and sea in tropical and subtropical latitudes, where they exist in conditions of high salinity, extreme tides, strong winds, high temperatures, and muddy, anaerobic soils. Rhizophoraceae is a key mangrove family, with highly developed morphological and physiological adaptations to extreme conditions. It is an ideal system for the study of the origin and adaptive evolution of mangrove plants. In this study, we characterized and comprehensively compared the transcriptomes of four mangrove species, from all four mangrove genera, as well as their closest terrestrial relative in Rhizophoraceae, using RNA-Seq. We obtained 41,936-48,845 unigenes with N50 values of 982-1,185 bp and 61.42-69.48% annotated for the five species in Rhizophoraceae. Orthology annotations of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Clusters of Orthologous Groups revealed overall similarities in the transcriptome profiles among the five species, whereas enrichment analysis identified remarkable genomic characteristics that are conserved across the four mangrove species but differ from their terrestrial relative. Based on 1,816 identified orthologs, phylogeny analysis and divergence time estimation revealed a single origin for mangrove species in Rhizophoraceae, which diverged from the terrestrial lineage ~56.4 million years ago (Mya), suggesting that the transgression during the Paleocene-Eocene Thermal Maximum may have been responsible for the entry of the mangrove lineage of Rhizophoraceae into intertidal environments. Evidence showed that the ancestor of Rhizophoraceae may have experienced a whole genome duplication event ~74.6 Mya, which may have increased the adaptability and survival chances of Rhizophoraceae during and following the Cretaceous-Tertiary extinction. The analysis of positive selection identified 10 positively selected genes from the ancestor branch of Rhizophoraceae mangroves, which were mainly associated with stress response, embryo development, and regulation of gene expression. Positive selection of these genes may be crucial for increasing the capability of stress tolerance (i.e., defense against salt and oxidative stress) and development of adaptive traits (i.e., vivipary) of Rhizophoraceae mangroves, and thus plays an important role in their adaptation to the stressful intertidal environments.

Preparative separation of gallocatechin gallate from Camellia ptilophylla using macroporous resins followed by sephadex LH-20 column chromatography.

Gallocatechin gallate (GCG) possesses multiple potential biological activities. However, the content of GCG in traditional green tea is too low which limits its in-depth pharmacological research and application. In the present study, a simple, efficient and environment-friendly chromatographic separation method was developed for preparative enrichment and separation of GCG from cocoa tea (Camellia ptilophylla) which contains high content of GCG. In the first step, the adsorption properties of selected resins were evaluated, and XAD-7HP resin was chosen by its adsorption and desorption properties for GCG. In order to maximize column efficiency for GCG collection, the operating parameters (e.g., flow rate, ethanol concentration, and bed height) were optimized. We found that the best combination was the feed concentration at 20mg/mL, flow rate at 0.75 BV/h and the ratio of diameter to bed heights as 1:12. Under these conditions, the purity of GCG was 45% with a recovery of 89%. In order to obtain pure target, a second step was established using column chromatography with sephadex LH-20 gel and 55% ethanol-water solution as eluent. After this step, the purity of the GCG was 91% with a recovery of 68% finally.

Development of microsatellite markers for Carallia brachiata (Rhizophoraceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Carallia brachiata to assess the genetic diversity and structure of this terrestrial species of the Rhizophoraceae. METHODS AND RESULTS: Based on transcriptome data for C. brachiata, 40 primer pairs were initially designed and tested, of which 18 were successfully amplified and 11 were polymorphic. For these microsatellites, one to three alleles per locus were identified. The observed and expected heterozygosities ranged from 0 to 0.727 and 0 to 0.520, respectively. In addition, all primers were successfully amplified in two congeners: C. pectinifolia and C. garciniifolia. CONCLUSIONS: The microsatellite markers described here will be useful in population genetic studies of C. brachiata and related species, suggesting that developing microsatellite markers from next-generation sequencing data can be efficient for genetic studies across this genus.

Inhibitory effects of cocoa tea (Camellia ptilophylla) in human hepatocellular carcinoma HepG2 in vitro and in vivo through apoptosis.

Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy beverage in southern China, has been recently found to be a potential candidate for the treatment of different diseases, including obesity and cancers. The present study aimed to evaluate the anti-liver cancer activities of green cocoa tea infusion (GCTI) in vitro and in vivo using human hepatocarcinoma cell line HepG2 cells and nude mice xenograft model. The apoptotic activities of GCTI were assessed using flow cytometry, Western blotting and immunohistochemical analysis. Our results showed that GCTI significantly inhibited the proliferation of HepG2 cells in a dose-dependent manner (IC50 values=292 µg/ml at 72 h). GCTI induced HepG2 cells to undergo apoptosis, which was demonstrated by cell cycle analysis and annexin-V and propidium iodide staining. The caspase cascade was activated as shown by significant proteolytic cleavage of caspase-3 and PARP in GCTI-treated cells in a dose- and time-dependent manner. In addition, GCTI increased the expression of cell cycle inhibitory proteins (p21, p27 and p53) and the Bax-to-Bcl-2 ratio to induce apoptosis. The antiproliferative effect of GCTI was confirmed in HepG2 xenograft nude mice. The tumor growth was effectively inhibited by GCTI in a dose-dependent manner as indicated by the decrease in tumor volume and tumor weight after 4 weeks of treatment. Administration of GCTI increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3-positive cells in the tumor section. In conclusion, these results revealed that GCTI may be a potential and promising agent of natural resource to treat liver cancer.