Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Genomic signals of local adaptation and hybridization in Asian white birch.

Disentangling the numerous processes that affect patterns of genome-wide diversity in widespread tree species has important implications for taxonomy, conservation, and forestry. Here, we investigate the population genomic structure of Asian white birch (Betula platyphylla) in China and seek to explain it in terms of hybridization, demography and adaptation. We generate whole genome sequence data from 83 individuals across the species range in China. Combining this with an existing data set for 79 European and Russian white birches, we show a clear distinction between B. pendula and B. platyphylla, which have sometimes been lumped taxonomically. Genomic diversity of B. platyphylla in north-western China and Central Russia is affected greatly by hybridization with B. pendula. Excluding these hybridized populations, B. platyphylla in China has a linear distribution from north-eastern to south-western China, along the edge of the inland mountainous region. Within this distribution, three genetic clusters are found, which we model as long diverged with subsequent episodes of gene flow. Patterns of covariation between allele frequencies and environmental variables in B. platyphylla suggest the role of natural selection in the distribution of diversity at 7609 SNPs of which 3767 were significantly differentiated among the genetic clusters. The putative adaptive SNPs are distributed throughout the genome and span 1633 genic regions. Of these genic regions, 87 were previously identified as candidates for selective sweeps in Eurasian B. pendula. We use the 7609 environmentally associated SNPs to estimate the risk of nonadaptedness for each sequenced B. platyphylla individual under a scenario of future climate change, highlighting areas where populations may be under future threat from rising temperatures.

Molecular and morphological analyses clarify species delimitation in section Costatae and reveal Betula buggsii sp. nov. (sect. Costatae, Betulaceae) in China.

BACKGROUND AND AIMS: Delineating closely related and morphologically similar species is difficult. Here, we integrate morphology, genetics, ploidy and geography to resolve species and subspecies boundaries in four trees of section Costatae (genus Betula): Betula ashburneri, B. costata, B. ermanii and B. utilis, as well as multiple subspecies and polyploid races. METHODS: We genotyped 371 individuals (20-133 per species) from 51 populations at 15 microsatellite markers, as well as a subset of individuals, using restriction-site associated DNA sequencing and nuclear internal transcribed spacers. We determined the ploidy level of eight individuals using flow cytometry and characterized leaf variation for a subset of 109 individuals by morphometric analysis. KEY RESULTS: Integration of multiple lines of evidence suggested a series of revisions to the taxonomy of section Costatae. Betula costata and B. ermanii were found to be valid. Molecular and leaf morphology analyses revealed little differentiation between diploid B. albosinensis and some samples of B. utilis ssp. utilis. By contrast, other B. utilis ssp. utilis samples and ssp. albosinensis formed a morphological continuum but differed based on genetics. Specifically, B. utilis ssp. albosinensis was divided into two groups with group I genetically similar to B. utilis ssp. utilis and group II, a distinct cluster, proposed as the new diploid species Betula buggsii sp. nov. Phylogenomic analysis based on 2285 620 single nucleotide polymorphisms identified a well-supported monophyletic clade of B. buggsii. Morphologically, B. buggsii is characterized by elongated lenticels and a distinct pattern of bark peeling and may be geographically restricted to the Qinling-Daba Mountains. CONCLUSIONS: Our integrated approach identifies six taxa within section Costatae: B. ashburneri, B. buggsii, B. costata, B. utilis ssp. utilis, B. utilis ssp. albosinensis and B. ermanii. Our research demonstrates the value of an integrative approach using morphological, geographical, genetic and ploidy-level data for species delineation.

A basic/helix-loop-helix transcription factor controls leaf shape by regulating auxin signaling in apple.

Climate-driven phenological change across local spatial gradients leads to leaf shape variation. At higher elevations, leaves of broadleaf species tend to become narrower, but the underlying molecular mechanism is largely unknown. In this study, a series of morphometric analyses and biochemical assays, combined with functional identification in apple, were performed. We show that the decrease in apple leaf width with increasing altitude is controlled by a basic/helix-loop-helix transcription factor (bHLH TF), MdbHLH3. The MdbHLH3-overexpressing lines have a lower transcript abundance of MdPIN1 encoding an auxin efflux carrier but a higher transcript abundance of MdGH3-2 encoding a putative auxin amido conjugate synthase, resulting in a lower free auxin concentration; feeding the transgenic leaves with exogenous auxin partially restores leaf width. MdbHLH3 transcriptionally suppresses and activates MdPIN1 and MdGH3-2, respectively, by specifically binding to their promoters. This alters auxin homeostasis and transport, consequently leading to changes in leaf shape. These findings suggest that the bHLH TF MdbHLH3 directly modulates auxin signaling in controlling leaf shape in response to local spatial gradients in apple.

An optimal mRNA marker for OSNA (One-step nucleic acid amplification) based lymph node metastasis detection in colorectal cancer patients.

BACKGROUND: We previously reported that the one-step nucleic acid amplification assay is effective for lymph node metastasis detection in breast cancer patients. This paper describes the identification of CK19 mRNA as an optimal marker and its cut-off value for use in the detection of one-step nucleic acid amplification-based lymph node metastasis in colorectal cancer patients. METHODS: Candidate mRNA markers selected from the genome-wide expressed sequence tag database were evaluated by quantitative RT-PCR using a mixture of metastasis-positive and another mixture of metastasis-negative lymph nodes (n = 5 each), followed by quantitative RT-PCR using metastasis-positive and -negative lymph nodes (n = 10 each) from 20 patients. The one-step nucleic acid amplification assay for mRNA markers selected above was examined using 28 positive lymph nodes from 19 patients and 38 negative lymph nodes from the 11 pN0 patients. RESULTS: Quantitative RT-PCR analyses of the 98 mRNAs selected from the genome-wide expressed sequence tag database and the subsequent quantitative RT-PCR analyses of the nine mRNAs selected above indicated that CK19 and CEA mRNAs have the highest capability for distinguishing between positive and negative lymph nodes. CK19, CEA and CK20 mRNAs were evaluated by the one-step nucleic acid amplification assay. An area under a receiver-operating-characteristic curve for CK19 mRNA (0.999) was slightly larger than that for CEA mRNA (0.946; P = 0.062) and significantly larger that than for CK20 mRNA (0.875; P = 0.006). CONCLUSION: We found that CK19 mRNA has the best diagnostic performance and its cut-off value for discriminating positive from negative lymph nodes can be set in the range of 75-500 copies/µl with 96.4% sensitivity and 100% specificity.

Betula mcallisteri sp. nov. (sect. Acuminatae, Betulaceae), a new diploid species overlooked in the wild and in cultivation, and its relation to the widespread B. luminifera.

Taxa are traditionally identified using morphological proxies for groups of evolutionarily isolated populations. These proxies are common characters deemed by taxonomists as significant. However, there is no general rule on which character or sets of characters are appropriate to circumscribe taxa, leading to discussions and uncertainty. Birch species are notoriously hard to identify due to strong morphological variability and factors such as hybridization and the existence of several ploidy levels. Here, we present evidence for an evolutionarily isolated line of birches from China that are not distinguishable by traditionally assumed taxon recognition proxies, such as fruit or leaf characters. We have discovered that some wild material in China and some cultivated in the Royal Botanic Gardens Edinburgh, formerly recognized as Betula luminifera, differ from other individuals by having a peeling bark and a lack of cambial fragrance. We use restriction site-associated DNA sequencing and flow cytometry to study the evolutionary status of the unidentified Betula samples to assess the extent of hybridization between the unidentified Betula samples and typical B. luminifera in natural populations. Molecular analyses show the unidentified Betula samples as a distinct lineage and reveal very little genetic admixture between the unidentified samples and B. luminifera. This may also be facilitated by the finding that B. luminifera is tetraploid, while the unidentified samples turned out to be diploid. We therefore conclude that the samples represent a yet unrecognized species, which is here described as Betula mcallisteri.

Preparation strategy and evaluation method of durable superhydrophobic rubber composites.

Superhydrophobic rubber composites have broad application prospects in national defense, industrial and agricultural production and daily life due to their special surface wettability. However, its poor durability at present seriously limits its practical application. Microstructure and low surface energy substances are the decisive factors to realize superhydrophobic surface. Therefore, three strategies to improve the durability of superhydrophobic surface were put forward, including improving the mechanical strength of microstructure, enhancing the adhesion between coating and substrate, and constructing self-repairing surface. On this basis, the preparation techniques of durable superhydrophobic rubber composites were summarized, and then the evaluation methods of durability of superhydrophobic rubber composites were introduced in detail from mechanical durability and chemical durability.

Three-dimensional tomographic microscopy technique with multi-frequency combination with partially coherent illuminations.

We demonstrate a three-dimensional (3D) optical diffraction tomographic technique with multi-frequency combination (MFC-ODT) for the 3D quantitative phase imaging of unlabeled specimens. Three sets of through-focus intensity images are captured under an annular aperture and two circular apertures with different coherence parameters. The 3D phase optical transfer functions (POTF) corresponding to different illumination apertures are combined to obtain a synthesized frequency response, achieving high-quality, low-noise 3D reconstructions with imaging resolution up to the incoherent diffraction limit. Besides, the expression of 3D POTF for arbitrary illumination pupils is derived and analyzed, and the 3D imaging performance of annular illumination is explored. It is shown that the phase-contrast washout effect in high-NA circular apertures can be effectively addressed by introducing a complementary annular aperture, which strongly boosts the phase contrast and improves the imaging resolution. By incorporating high-NA illumination as well as high-NA detection, MFC-ODT can achieve a theoretical transverse resolution up to 200 nm and an axial resolution of 645 nm. To test the feasibility of the proposed MFC-ODT technique, the 3D refractive index reconstruction results are based on a simulated 3D resolution target and experimental investigations of micro polystyrene bead and unstained biological samples are presented. Due to its capability for high-resolution 3D phase imaging as well as the compatibility with a widely available commercial microscope, the MFC-ODT is expected to find versatile applications in biological and biomedical research.

Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress.

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. In this study, we have investigated the role of Syk in p38 activation and the regulation of cell-cycle progression upon oxidative stress. In B cells, p38 is activated by hydrogen peroxide (H(2)O(2)) stimulation. Syk is required for p38 activation following stimulation with 10-100 microM H(2)O(2), but not with 1 mM H(2)O(2). H(2)O(2)-induced p38 activation is abrogated in phospholipase C-gamma2 (PLC-gamma2)-deficient as well as Syk-deficient cells, suggesting that Syk activates p38 through PLC-gamma2 upon H(2)O(2) stimulation. Although stimulation with 20-100 microM H(2)O(2) induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H(2)O(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 microM H(2)O(2) exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H(2)O(2)-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H(2)O(2) stimulation. These findings indicate that Syk plays a role in the regulation of cell-cycle progression in G2/M phase via p38-dependent and -independent pathways after oxidative stress.

An accurate and rapid detection of lymph node metastasis in non-small cell lung cancer patients based on one-step nucleic acid amplification assay.

A sublobar resection is currently recognized as an option for early small-sized non-small cell lung cancer (NSCLC), and intraoperative rapid and accurate lymph node assessment is required for a complete resection. To solve this issue, we investigated the clinical utility of one-step nucleic acid amplification (OSNA) assay, an automated rapid molecular diagnostic method and its optimal mRNA marker for detection of lymph node metastasis in lung cancer. We extracted 16 target candidate mRNA markers with high expression in lung cancer from a genetic database, and then quantified their expression levels by quantitative RT-PCR using surgically dissected lymph nodes with or without metastasis. Cytokeratin 19 (CK19), cytokeratin 7 (CK7), stratifin (SFN), and anterior gradient homolog 2 (AGR2) showed significant differences for mRNA expression between metastasis-negative and -positive lymph nodes in quantitative-RT-PCR screening. CK19 and CK7 were finally selected as potential target markers and were quantified using OSNA assay findings of 165 dissected lymph nodes obtained from 49 lung cancer patients. The OSNA assay with CK19 and CK7 were completed within 40 min and their positive predictive value, negative predictive value, and accuracy comparing to pathological diagnosis with hematoxylin-eosin staining and immunohistochemistry were shown to be 95.0%, 99.3%, and 98.8%, and 85.0%, 97.9%, and 96.4%, respectively, using a cut-off value of 250 copies/µL. Among the 165 lymph nodes tested, 1 false negative result was due to massive necrosis of cancer cells and 1 false positive was caused by the allocation bias of cancer cells in the sampling in patient with pleural dissemination. The best performance was observed when CK19 was used as a marker, while the addition of CK7 mRNA as a marker did not increase sensitivity or specificity. In conclusion, an OSNA assay using CK19 could be effective for molecular diagnosis of lymph node metastasis in lung cancer. This is the first report suggesting the potential clinical utility of OSNA assay for intraoperative rapid diagnosis of nodal status in lung cancer.