Near-Infrared Perylenecarboximide Fluorophores for Live-Cell Super-Resolution Imaging.

Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N3) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

The complete chloroplast genome sequence of Chrysojasminum subhumile and its phylogenetic position within Oleaceae.

We assembled and characterized the complete chloroplast genome sequence of Chrysojasminum subhumile (W.W.Sm.) Banfi & Galasso 2014, a valuable horticultural and medicinal plant species. The total genome size was 159,918 bp in length and the GC content was 37.4%. It displayed a circular structure and could be divided into a large single-copy region, a small single-copy region, and a pair of inverted repeat regions. The genome encoded a total of 131 unique genes, including 82 protein-coding genes, 41 tRNA genes, and eight rRNA genes. Among these genes, 17 contained a single intron, and two genes had two introns. Phylogenetic analysis results showed that C. subhumile was closely related to Jasminum.

Single Molecule Localization Microscopy for Studying Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are 30-200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

The complete chloroplast genome of Tulipa gesneriana (Liliaceae) and its phylogenetic analysis.

The complete chloroplast genome sequence of Tulipa gesneriana L. was determined to investigate its phylogenetic position. This plastome is 151,958 base pairs (bp) in length, and comprises two inverted repeat (IRa and IRb) regions of 26,352 bp, a small single-copy region of 17,123 bp and a large single-copy region of 82,131 bp. The GC contents of the cp genome were 36.6%. In total, we annotated 126 genes including 81 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis based on nine chloroplast genomes indicates that T. gesneriana is closely related to T. iliensis and T. thianschanica.

Shielding Unit Engineering of NIR-II Molecular Fluorophores for Improved Fluorescence Performance and Renal Excretion Ability.

Molecular fluorophores emitting in the second near-infrared (NIR-II) window with good renal excretion ability are favorable for in vivo bio-imaging and clinical applications. So far, renally excretable fluorophores are still less studied. Understanding the influences of molecular structure on optical properties and renal excretion abilities are vital for fluorophore optimization. Herein, a series of shielding unit-donor-acceptor-donor-shielding unit (S-D-A-D-S) NIR-II molecular fluorophores are designed and synthesized with dialkoxy chains substituted benzene as the S unit. The anchoring positions of dialkoxy chains on benzene are tuned as meso-2,6, para-2,5, or ortho-3,4 to afford three fluorophores: BGM6P, BGP6P and BGO6P, respectively. Experimental and calculation results reveal that alkoxy side chains anchored closer to the conjugated backbone can provide better protection from water molecules and PEG chains, affording higher fluorescence quantum yield (QY) in aqueous solutions. Further, these side chains can enable good encapsulation of backbone, resulting in decreased binding with albumin and improved renal excretion. Thus, fluorophore BGM6P with meso-2,6-dialkoxy chains exhibits the highest quantum yield and fastest renal excretion. This work emphasizes the important roles of side chain patterns on optimizing NIR-II fluorophores with high brightness and renal excretion ability.

The complete chloroplast genome sequence of Acorus gramineus (Acoraceae).

The complete chloroplast genome sequence of Acorus gramineus was assembled and characterized as a resource for future genetic studies. With a total length of 152,887 bp, the chloroplast genome comprised of a large single-copy (LSC) region of 83,005 bp, a small single-copy (SSC) region of 18,230 bp, and two inverted repeat (IR) regions of 25,826 bp. The overall GC contents of the chloroplast genome were 38.7%. A total of 115 genes were predicted, consisting of 80 protein-coding genes, 31 tRNA genes, and 4 rRNA genes. In these genes, nine genes contained one intron and two genes contained two introns. Phylogenetic analysis confirmed the position of A. gramineus within the monocots.

The complete chloroplast genome of Myxopyrum hainanense and phylogenic analysis of Oleaceae.

We assembled and characterized the complete chloroplast genome sequence of Myxopyrum hainanense to investigate its phylogenetic position. The plastome is 156,064 bp in length, which is comprised of a large single-copy (LSC) region of 86,851 bp, a small single-copy (SSC) region of 17,837 bp, and two inverted repeat (IR) regions of 25,688 bp. The overall GC content of the plastome was 37.7. The new sequence comprised total 135 genes, including 87 protein-coding genes, 8 ribosomal RNA genes, and 40 tRNA genes. Phylogenetic analysis showed that M. hainanense was close to Nyctanthes arbor-tristis.

Genomic basis of homoploid hybrid speciation within chestnut trees.

Hybridization can drive speciation. We examine the hypothesis that Castanea henryi var. omeiensis is an evolutionary lineage that originated from hybridization between two near-sympatric diploid taxa, C. henryi var. henryi and C. mollissima. We produce a high-quality genome assembly for mollissima and characterize evolutionary relationships among related chestnut taxa. Our results show that C. henryi var. omeiensis has a mosaic genome but has accumulated divergence in all 12 chromosomes. We observe positive correlation between admixture proportions and recombination rates across the genome. Candidate barrier genomic regions, which isolate var. henryi and mollissima, are re-assorted in the hybrid lineage. We further find that the putative barrier segments concentrate in genomic regions with less recombination, suggesting that interaction between natural selection and recombination shapes the evolution of hybrid genomes during hybrid speciation. This study highlights that reassortment of parental barriers is an important mechanism in generating biodiversity.

Multiplexed NIR-II Probes for Lymph Node-Invaded Cancer Detection and Imaging-Guided Surgery.

Tumor-lymph node (LN) metastasis is the dominant prognostic factor for tumor staging and therapeutic decision-making. However, concurrently visualizing metastasis and performing imaging-guided lymph node surgery is challenging. Here, a multiplexed-near-infrared-II (NIR-II) in vivo imaging system using nonoverlapping NIR-II probes with markedly suppressed photon scattering and zero-autofluorescence is reported, which enables visualization of the metastatic tumor and the tumor metastatic proximal LNs resection. A bright and tumor-seeking donor-acceptor-donor (D-A-D) dye, IR-FD, is screened for primary/metastatic tumor imaging in the NIR-IIa (1100-1300 nm) window. This optimized D-A-D dye exhibits greatly improved quantum yield of organic D-A-D fluorophores in aqueous solutions (≈6.0%) and good in vivo performance. Ultrabright PbS/CdS core/shell quantum dots (QDs) with dense polymer coating are used to visualize cancer-invaded sentinel LNs in the NIR-IIb (>1500 nm) window. Compared to clinically used indocyanine green, the QDs show superior brightness and photostability (no obvious bleaching even after continuous laser irradiation for 5 h); thus, only a picomolar dose is required for sentinel LNs detection. This combination of dual-NIR-II image-guided surgery can be performed under bright light, adding to its convenience and appeal in clinical use.

Variation in the degree of reciprocal herkogamy affects the degree of legitimate pollination in a distylous species.

Distyly is a widespread floral polymorphism characterized by the flowers within a population showing reciprocal placement of the anthers and stigma. Darwin hypothesizes that distyly evolves to promote precise pollen transfer between morphs. Primula chungensis exhibits two types of anther heights, and these two types of anthers show pollen of two different size classes. To understand whether the stigma could capture more pollen grains from the anthers of the pollen donor as the separation between the stigma of pollen receiver and the anther of pollen donor decreased, the present research assessed the source of the pollen load in a series of open-pollinated flowers with continuous variation of style lengths. Individuals with continuous variation of style length were tagged, and the selected flowers in the tagged plants were emasculated the day before dehiscence. The stigma of the emasculated flowers was fixed in fuchsin gel at the end of blooming. We assessed the pollen sources on each stigma by taking photos under a microscope and measured the diameter of each conspecific pollen grain with ImageJ. We found that a shorter distance from the stigmas to the anthers of a pollen donor gave the flower a higher capacity to receive pollen from those anthers. Our result provides a new evidence that distyly could promote the pollen transfer between morphs, which is consistent with Darwin's hypothesis of disassortative pollination. An alternative hypothesis for the evolution of distyly (e.g. selfing avoidance) might also be true, but less likely, because self-incompatibility would greatly avoid self-fertilization for many distylous species.

Correlation between the timing of autonomous selfing and floral traits: a comparative study from three selfing Gentianopsis species (Gentianaceae).

About 20% of angiosperms employ self-fertilization as their main mating strategy. In this study, we aimed to examine how the selfing timing correlated with floral traits in three Gentianopsis species in which autonomous selfing is achieved through filament elongation. Although the three Gentianopsis species exhibit no significant variation in their capacity for autonomous selfing, flowers of G. grandis last longer, are larger and have a higher corolla biomass, P/O ratios and male biomass allocation than those of G. paludosa, and especially those of G. contorta. Autonomous selfing occurs in the early floral life of G. paludosa and G. contorta and in the later floral life of G. grandis. Seed production mainly results from autonomous selfing in G. paludosa and G. contorta; however, G. grandis could be more described as having a mixed mating system. We suggest that autonomous selfing in later floral life increases the chance of cross-pollination prior to this, while autonomous selfing in early floral life offers a selective advantage to plants by reducing the resource investment in traits that may increase pollinator attraction and visitation.

Natural hybridization and reproductive isolation between two Primula species.

Natural hybridization frequently occurs in plants and can facilitate gene flow between species, possibly resulting in species refusion. However, various reproductive barriers block the formation of hybrids and maintain species integrity. Here, we conducted a field survey to examine natural hybridization and reproductive isolation (RI) between sympatric populations of Primula secundiflora and P. poissonii using ten nuclear simple sequence repeat (SSR) loci. Although introgressive hybridization occurred, species boundaries between P. secundiflora and P. poissonii were maintained through nearly complete reproductive isolation. These interfertile species provide an excellent model for studying the RI mechanisms and evolutionary forces that maintain species boundaries.

Correction: Associative Mechanisms Allow for Social Learning and Cultural Transmission of String Pulling in an Insect.

[This corrects the article DOI: 10.1371/journal.pbio.1002564.].

Associative Mechanisms Allow for Social Learning and Cultural Transmission of String Pulling in an Insect.

Social insects make elaborate use of simple mechanisms to achieve seemingly complex behavior and may thus provide a unique resource to discover the basic cognitive elements required for culture, i.e., group-specific behaviors that spread from "innovators" to others in the group via social learning. We first explored whether bumblebees can learn a nonnatural object manipulation task by using string pulling to access a reward that was presented out of reach. Only a small minority "innovated" and solved the task spontaneously, but most bees were able to learn to pull a string when trained in a stepwise manner. In addition, naïve bees learnt the task by observing a trained demonstrator from a distance. Learning the behavior relied on a combination of simple associative mechanisms and trial-and-error learning and did not require "insight": naïve bees failed a "coiled-string experiment," in which they did not receive instant visual feedback of the target moving closer when tugging on the string. In cultural diffusion experiments, the skill spread rapidly from a single knowledgeable individual to the majority of a colony's foragers. We observed that there were several sequential sets ("generations") of learners, so that previously naïve observers could first acquire the technique by interacting with skilled individuals and, subsequently, themselves become demonstrators for the next "generation" of learners, so that the longevity of the skill in the population could outlast the lives of informed foragers. This suggests that, so long as animals have a basic toolkit of associative and motor learning processes, the key ingredients for the cultural spread of unusual skills are already in place and do not require sophisticated cognition.

Asymmetrical disassortative pollination in a distylous primrose: the complementary roles of bumblebee nectar robbers and syrphid flies.

Heterostyly is a floral polymorphism characterized by reciprocal herkogamy maintained through high levels of mating between morphs, serviced by appropriate pollinators. We studied how differential efficiency and abundance of distinct pollinators affect plant female reproduction in self- and intra-morph incompatible distylous Primula secundiflora. Bumblebees and syrphid flies were found to be the most abundant floral visitors. Bumblebees frequently exhibited nectar-robbing behavior. Because the robbing holes were always situated between the high- and low-level organs on both morphs, nectar-robbing bumblebees only pollinated S-styled flowers. L-styled flowers set four times as many seeds as did S-styled flowers after being visited by pollen-collecting syrphid flies. The natural female fecundity and the magnitude of pollen limitation varied between the morphs within populations because of the mosaic distribution of nectar-robbing bumblebees and syrphid flies. L-styled flowers and S-styled flowers set the same number of seeds after supplemental hand pollination, indicating equivalent female reproductive potential. We suggest that bumblebee nectar robbers and syrphid flies play an important role in sustaining the floral dimorphism of heterostyly in P. secundiflora because of their complementary roles in the pollination system.

Out of Africa: evidence of the obligate mutualism between long corolla tubed plant and long-tongued fly in the Himalayas.

Mutualism between long corolla tubed plants and their potential pollinators, long-tongued flies, is a classic example of coevolution, but to date, has only been reported from the regions of southern Africa. Many plant species from the Himalayas also show botanical characteristics that could be consistent with pollination by long-tongued flies. Here, we seek the evidence of the "long-tongued-long tubed fly/flower" mutualism out of Africa, in a different continent and climatic region, the Himalayas.Floral traits of Himalayan region endemic alpine genus, Roscoea, indicate possible mutualism with long-tongued flies for pollination success; however, effective pollinators of this genus are yet unknown. This study investigates whether long-tongued flies and Roscoea purpurea in Nepal Himalayas show exclusive mutualism for their survival/reproduction.We made extensive observations of floral visitors of R. purpurea and food source of Philoliche longirostris across their wide ranges of populations in Nepal Himalayas for three consecutive years (2012-2014). To confirm the obligate reliance of R. purpurea upon P. longirostris for pollination success, manipulated pollination experiments were conducted at two populations for 2 years. Similarly foraging behavior, visitation frequency, and pollination efficiency of P. longirostris were assessed at two populations for 2 years, and its contribution for the reproductive success of R. purpurea was evaluated. Our results indicate that R. purpurea is self-compatible but lacks autonomous selfing and obligatorily relies on P. longirostris for reproductive success. Across all populations, P. longirostris was observed as an exclusive and highly efficient pollinator of R. purpurea, while P. longirostris exclusively depends up on R. purpurea for food source.Out of Africa, this study provides the first evidence of long-tongued fly pollination system and indicates the possibility of additional instances of such a rare phenomenon in the Himalayas. Finding of specialized pollinator of Roscoea only at its evolutionary center indicates that Roscoea species are originally pollinated by long-tongued flies. Spatial mismatch with specialized pollinators may have induced the evolution of autonomous selfing in North Indochinese clades of Roscoea. This finding thus substantiates how geographic disjunction causes the shifting of pollination mechanism in closely related plant species.

Size-dependent gender modification in Lilium apertum (Liliaceae): does this species exhibit gender diphasy?

BACKGROUND AND AIMS: Variation in the relative female and male reproductive success of flowering plants is widespread, despite the fundamental hermaphroditic condition of the majority of species. In many hermaphroditic populations, environmental conditions and their influence on development and size can influence the gender expression of individuals through the formation of hermaphroditic and unisexual flowers. This study investigates the hypothesis that the bulbous, animal-pollinated, perennial Lilium apertum (Liliaceae) exhibits a form of size-dependent gender modification known as gender diphasy, in which the sexual expression of individuals depends on their size, with plants often changing sex between seasons. METHODS: Variation in floral traits was examined in relation to their size using marked individuals in natural populations, and also under glasshouse conditions. Measurements were taken of the height, flower number, floral sex expression, flower size, flower biomass and pollen production of individuals over consecutive years between 2009 and 2012 in seven populations in south-west China. KEY RESULTS: Flowers of L. apertum are either perfect (hermaphroditic) or staminate (male) and, in any given season, plants exhibit one of three sex phenotypes: only hermaphrodite flowers, a mixture of hermaphroditic and male flowers, or only male flowers. Transitions between each of these sex phenotypes were observed over consecutive years and were commonly size-dependent, particularly transitions from small plants bearing only male flowers to those that were taller with hermaphroditic flowers. Hermaphroditic flowers were significantly larger, heavier and produced more pollen than male flowers. CONCLUSIONS: The results for L. apertum are consistent with the 'size advantage hypothesis' developed for animal species with sex change. The theory predicts that when individuals are small they should exhibit the sex for which the costs of reproduction are less, and this usually involves the male phase. L. apertum provides an example of gender diphasy, a rare sexual system in flowering plants.

Nectar robbers pollinate flowers with sexual organs hidden within corollas in distylous Primula secundiflora (Primulaceae).

Nectar robbers are thought rarely to pollinate flowers, especially those with sexual organs hidden within corollas. In this study, we examined whether robbers pollinate flowers of distylous Primula secundiflora. Distylous plants have two floral morphs. Pin flowers have long styles and short stamens, and thrum flowers have short styles and long stamens. Flowers of P. secundiflora were commonly robbed by bumble-bees, and robbing holes were always situated between high and low sexual organs for both floral morphs. We observed that pollen grains of pin flowers were removed while thrum flowers received fresh pollen grains immediately after flowers were robbed. We manipulated flowers so that only nectar robbers could visit them. This resulted in 98 per cent of thrum flowers and 6 per cent of pin flowers setting fruit, and seed number per thrum fruit was also significantly higher than per pin fruit. Our findings suggest that nectar robbers transfer pollen from pin flowers to thrum flowers effectively, and consequently increase male fitness of the pin morph and female fitness of the thrum morph. Such asymmetrical pollen flow caused by nectar robbers may act as an important selective agent in floral fitness and evolution of distyly.

Decoration of multiwalled carbon nanotubes with CoO and NiO nanoparticles and studies of their magnetism properties.

Multiwalled carbon nanotubes (MWNTs) have been successfully decorated with magnetic CoO and NiO nanoparticles via the interaction of M(NH(3))x(2+) (M = Co,Ni) ions and the carboxyl groups on the surface of acid-treated MWNTs. The obtained composites were characterized by X-ray diffraction, scanning electron and transmission electron microscopies. The soaking time plays an important role in the preparation of the composites. Magnetic measurements indicate that both the CoO/MWNT and NiO/MWNT composites present superparamagnetic behavior which can be attributed to the small size of CoO and NiO nanoparticles.