High-density lipoprotein-biomimetic nanocarriers for glioblastoma-targeting delivery: the effect of shape.

Rational design of the physicochemical properties of nanocarriers can optimize their pharmacokinetics, biodistribution, intratumoral penetration and tumor bioavailability. In particular, particle shape is one of the crucial parameters that can impact the circulation time, tumor accumulation and tumor cell internalization of nanocarrier. Biomimetic reconstituted high-density lipoprotein (rHDL), by mimicking the endogenous shape and structure of high-density lipoprotein, has been indicated as a promising tumor-targeting nanoparticulate drug delivery system whereas the effect of shape on tumor-targeting efficiency has not been fully evaluated. Herein, we constructed apolipoprotein E-based biomimetic rHDL in both discoidal form (d-rHDL) and spherical form (s-rHDL), and compared their efficiency in glioblastoma multiforme (GBM)-targeting delivery. s-rHDL showed higher cellular association in GBM cells especially at a high exposure dosage or after a long incubation time. Moreover, it exhibited deeper penetration in 3D GBM spheroids in vitro and higher accumulation at the GBM site in vivo with the GBM-targeting accumulation of s-rHDL increased by 73% when compared with that of d-rHDL at 24 h post-injection. The findings collectively indicated that s-rHDL might serve as a more efficient nanocarrier for glioblastoma-targeting drug delivery.

High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes.

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially ßCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes.

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes.

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.

Isolation and characterization of 15 microsatellite markers from the spring orchid (Cymbidium goeringii) (Orchidaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the important and popular ornamental spring orchid, Cymbidium goeringii, to investigate its genetic diversity and population genetic structure. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 15 primer sets were identified in two wild populations. The number of alleles per locus ranged from 2 to 5, with a mean of 3.3. The observed and expected heterozygosities varied from 0.167 to 0.917 and from 0.159 to 0.822, respectively. All these primers successfully amplified in the congener C. tortisepalum, and 12 primers were found useful in C. faberi and C. sinense. CONCLUSIONS: These markers will facilitate further studies on the population genetics of Cymbidium goeringii and other congeneric species.

Allometry Between Vegetative and Reproductive Traits in Orchids.

In flowering plants, inflorescence characteristics influence both seed set and pollen contribution, while inflorescence and peduncle size can be correlated with biomass allocation to reproductive organs. Peduncles also play a role in water and nutrient supply of flowers, and mechanical support. However, it is currently unclear whether inflorescence size is correlated with peduncle size. Here, we tested whether orchids with large diameter peduncles bear more and larger flowers than those with smaller peduncles by analyzing 10 traits of inflorescence, flower, and leaf in 26 species. Peduncle diameters were positively correlated with inflorescence length and total floral area, indicating that species with larger peduncles tended to have larger inflorescences and larger flowers. We also found strongly positive correlation between inflorescence length and leaf area, and between total floral area and total leaf area, which suggested that reproductive organs may be allometrically coordinated with vegetative organs. However, neither flower number nor floral dry mass per unit area were correlated with leaf number or leaf dry mass per unit area, implying that the function between leaf and flower was uncoupled. Our findings provided a new insight for understanding the evolution of orchids, and for horticulturalists interested in improving floral and inflorescence traits in orchids.

Photosystem I is tolerant to fluctuating light under moderate heat stress in two orchids Dendrobium officinale and Bletilla striata.

Under natural field conditions, plants usually experience fluctuating light (FL) under moderate heat stress in summer. However, responses of photosystems I and II (PSI and PSII) to such combined stresses are not well known. Furthermore, the role of water-water cycle (WWC) in photoprotection in FL under moderate heat stress is poorly understood. In this study, we examined chlorophyll fluorescence and P700 redox state in FL at 42 °C in two orchids, Dendrobium officinale (with high WWC activity) and Bletilla striata (with low WWC activity). After FL treatment at 42 °C, PSI activity maintained stable while PSII activity decreased significantly in these two orchids. In D. officinale, the WWC could rapidly consume the excess excitation energy in PSI and thus avoided an over-reduction of PSI upon any increase in illumination. Therefore, in D. officinale, WWC likely protected PSI in FL at 42 °C. In B. striata, heat-induced PSII photoinhibition down-regulated electron flow from PSII and thus prevented an over-reduction of PSI after transition from low to high light. Consequently, in B. striata moderate PSII photoinhibition could protected PSI in FL at 42 °C. We conclude that, in addition to cyclic electron flow, WWC and PSII photoinhibition-repair cycle are two important strategies for preventing PSI photoinhibition in FL under moderate heat stress.

Floral Longevity of Paphiopedilum and Cypripedium Is Associated With Floral Morphology.

Floral longevity (FL) is an important trait influencing plant reproductive success by affecting the chance of insect pollination. However, it is still unclear which factors affect FL, and whether FL is evolutionarily associated with structural traits. Since construction costs and water loss by transpiration play a role in leaf longevity, we speculated that floral structures may affect the maintenance and loss of water in flowers and, therefore, FL. Here, we investigated the slipper orchid Paphiopedilum and Cypripedium, which are closely related, but strongly differ in their FL. To understand the evolutionary association of floral anatomical traits with FL, we used a phylogenetic independent comparative method to examine the relationships between 30 floral anatomical traits and FL in 18 species of Paphiopedilum and Cypripedium. Compared with Paphiopedilum species, Cypripedium species have lower values for floral traits related to drought tolerance and water retention capacity. Long FL was basically accompanied by the thicker epidermal and endodermal tissues of the floral stem, the thicker adaxial and abaxial epidermis of the flower, and low floral vein and stomatal densities. Vein density of the dorsal sepals and synsepals was negatively correlated with stomatal density. Our results supported the hypothesis that there was a correlation between FL and floral anatomical traits in slipper orchids. The ability to retain water in the flowers was associated with FL. These findings provide a new insight into the evolutionary association of floral traits with transpirational water loss for orchids under natural selection.

Mycorrhizal specificity, preference, and plasticity of six slipper orchids from South Western China.

Mycorrhizal fungi of six endangered species, Paphiopedilum micranthum, Paphiopedilum armeniacum, Paphiopedilum dianthum, Cypripedium flavum, Cypripedium guttatum, and Cypripedium tibeticum, from two closely related genera in the Orchidaceae from Southwestern China, were characterized using the nuclear internal transcribed spacer (ITS) and part of the large subunit gene of mitochondrial rDNA (mtLSU) sequences. The most frequently detected fungi belonged to the Tulasnellaceae. These fungi were represented by 25 ITS sequence types and clustered into seven major clades in the phylogenetic analysis of 5.8S sequences. Species of Paphiopedilum and Cypripedium shared no fungal ITS sequence types in common, but their fungal taxa sometimes occurred in the same major clade of the 5.8S phylogenetic tree. Although it had several associated fungal ITS sequence types in a studied plot, each orchid species had in general only a single dominant type. The fungal sequence type spectra of different species of Paphiopedilum from similar habitats sometimes overlapped; however, the dominant sequence types differed among the species and so did the sequence-type spectra within Cypripedium. Orchids of P. micranthum and P. armeniacum transplanted from the field and grown in two greenhouses had a greater number of mycorrhizal associations than those sampled directly from the field. Root specimens from P. micranthum taken from the greenhouses were preferably associated with mycobionts of the Tulasnella calospora complex, while those from the field had mycorrhizal associations of other tulasnelloid taxa. Such plasticity in mycorrhizal associations makes ex situ conservation or even propagation by means of mycorrhization of axenically grown seedlings possible.

Correlated evolution of leaf and root anatomic traits in Dendrobium (Orchidaceae).

The whole-plant economic spectrum concept predicts that leaf and root traits evolve in coordination to cope with environmental stresses. However, this hypothesis is difficult to test in many species because their leaves and roots are exposed to different environments, above- and below-ground. In epiphytes, both leaves and roots are exposed to the atmosphere. Thus, we suspect there are consistent water conservation strategies in leaf and root traits of epiphytes due to similar selection pressures. Here, we measured the functional traits of 21 species in the genus Dendrobium, which is one of the largest epiphytic taxa in the family Orchidaceae, and used phylogenetically independent contrasts to test the relationships among traits, and between traits and the environment. Our results demonstrate that species with a thicker velamen tended to have thicker roots, a thicker root cortex and vascular cylinder, and a larger number of vessels in the root. Correspondingly, these species also had higher leaf mass per area, and thicker leaf lower cuticles. Leaf and root traits associated with water conservation showed significantly positive relationships. The number of velamen layers, leaf density and the ratio of vascular cylinder radius to root radius were significantly affected by the species' differing environments. Thus, traits related to water conservation and transport may play an important role in helping Dendrobium cope with the cool and dry conditions found at high elevations. These findings confirmed the hypothesis that leaf and root traits have evolved in coordination, and also provide insights into trait evolution and ecological adaptation in epiphytic orchids.

Lipid-coated calcium phosphate nanoparticle and beyond: a versatile platform for drug delivery.

In recent years, lipid-coated calcium-phosphate (LCP) nanoparticle has been developed as a versatile platform for delivery of various therapeutics including gene, protein/peptide, chemotherapeutics and theranostic agents. The high endosomal escape, coupled with the ability to efficiently encapsulate phosphorylated drugs or prodrugs, make LCP become attractive vehicle for drug delivery. Additionally, the principle behind LCP formulation has also allowed rational design of LCP-derived nanoparticles (NPs) with other solid core or lipid membrane to overcome the various drug delivery barriers. Here, we briefly review the history of the development of LCP NPs, highlight the optimisations and modulations in the development process, and summarise the major applications of LCP NPs and LCP-derived NPs in drug delivery.

Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells via macropinocytosis.

Hyperactivated Ras regulates many oncogenic pathways in several malignant human cancers including glioblastoma and it is an attractive target for cancer therapies. Ras activation in cancer cells drives protein internalization via macropinocytosis as a key nutrient-gaining process. By utilizing this unique endocytosis pathway, here we create a biologically inspired nanostructure that can induce cancer cells to 'drink drugs' for targeting activating transcription factor-5 (ATF5), an overexpressed anti-apoptotic transcription factor in glioblastoma. Apolipoprotein E3-reconstituted high-density lipoprotein is used to encapsulate the siRNA-loaded calcium phosphate core and facilitate it to penetrate the blood-brain barrier, thus targeting the glioblastoma cells in a macropinocytosis-dependent manner. The nanostructure carrying ATF5 siRNA exerts remarkable RNA-interfering efficiency, increases glioblastoma cell apoptosis and inhibits tumour cell growth both in vitro and in xenograft tumour models. This strategy of targeting the macropinocytosis caused by Ras activation provides a nanoparticle-based approach for precision therapy in glioblastoma and other Ras-activated cancers.

Trait evolution in the slipper orchid paphiopedilum (Orchidaceae) in China.

The well-known orchid genus Paphiopedilum has attracted much attention from biologists because of its diverse floral traits. Although these traits have been thoroughly described, little is known about their evolutionary trajectory. In this study, we explored their evolutionary patterns and trajectory through phylogenetic analyses and close observations, and 10 characters in 21 Chinese species mapped onto an existing phylogenetic tree. Lip shape, staminode shape, petal shape, and petal width are relatively congruent with molecular phylogenies, thereby validating the existing traditional classification system. All four of those characters, along with flower number, are strongly conserved, and are significantly affected by phylogeny. By contrast, flower color (including that of the dorsal sepal, lip, and petal) is significantly convergent among those examined species and less affected by phylogeny. Therefore, this character is independent of evolution and mainly influenced by environmental factors. All of these characters are key, classical indicators when distinguishing among species within the subgenera Brachypetalum and Paphiopedilum.

Orchid Species Richness along Elevational and Environmental Gradients in Yunnan, China.

The family Orchidaceae is not only one of the most diverse families of flowering plants, but also one of the most endangered plant taxa. Therefore, understanding how its species richness varies along geographical and environmental gradients is essential for conservation efforts. However, such knowledge is rarely available, especially on a large scale. We used a database extracted from herbarium records to investigate the relationships between orchid species richness and elevation, and to examine how elevational diversity in Yunnan Province, China, might be explained by mid-domain effect (MDE), species-area relationship (SAR), water-energy dynamics (WED), Rapoport's Rule, and climatic variables. This particular location was selected because it is one of the primary centers of distribution for orchids. We recorded 691 species that span 127 genera and account for 88.59% of all confirmed orchid species in Yunnan. Species richness, estimated at 200-m intervals along a slope, was closely correlated with elevation, peaking at 1395 to 1723 m. The elevational pattern of orchid richness was considerably shaped by MDE, SAR, WED, and climate. Among those four predictors, climate was the strongest while MDE was the weakest for predicting the elevational pattern of orchid richness. Species richness showed parabolic responses to mean annual temperature (MAT) and mean annual precipitation (MAP), with maximum richness values recorded at 13.7 to 17.7°C for MAT and 1237 to 1414 mm for MAP. Rapoport's Rule also helped to explain the elevational pattern of species richness in Yunnan, but those influences were not entirely uniform across all methods. These results suggested that the elevational pattern of orchid species richness in Yunnan is collectively shaped by several mechanisms related to geometric constraints, size of the land area, and environments. Because of the dominant role of climate in determining orchid richness, our findings may contribute to a better understanding of the potential effects of climate change on orchid diversity, and the development of conservation strategies for orchids.

The effect of neat ionic liquid on the folding of short peptides.

Using circular dichroism spectroscopy, we show evidence of unusual folding behaviour for several designed peptides in neat ionic liquid. Helical peptides, AKA(2) and Trp-cage, exhibit heat-induced folding, with stable helical structure persisting to 96 °C, whereas the ß-hairpin Trpzip4 is destabilized by the neat [C(4)mpy][Tf(2)N].