Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine ...(PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.
A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide ...nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.
Germline determination is essential for species survival and evolution in multicellular organisms. In most flowering plants, formation of the female germline is initiated with specification of one ...megaspore mother cell (MMC) in each ovule; however, the molecular mechanism underlying this key event remains unclear. Here we report that spatially restricted auxin signaling promotes MMC fate in Arabidopsis. Our results show that the microRNA160 (miR160) targeted gene ARF17 (AUXIN RESPONSE FACTOR17) is required for promoting MMC specification by genetically interacting with the SPL/NZZ (SPOROCYTELESS/NOZZLE) gene. Alterations of auxin signaling cause formation of supernumerary MMCs in an ARF17- and SPL/NZZ-dependent manner. Furthermore, miR160 and ARF17 are indispensable for attaining a normal auxin maximum at the ovule apex via modulating the expression domain of PIN1 (PIN-FORMED1) auxin transporter. Our findings elucidate the mechanism by which auxin signaling promotes the acquisition of female germline cell fate in plants.
MicroRNAs (miRNAs) have emerged as key regulators of gene expression at the post-transcriptional level in both plants and animals. However, the specific functions of MIRNAs (MIRs) and the mechanisms ...regulating their expression are not fully understood. Previous studies showed that miR160 negatively regulates three genes that encode AUXIN RESPONSE FACTORs (ARF10, -16, and -17). Here, we characterized floral organs in carpels (foc), an Arabidopsis mutant with a Ds transposon insertion in the 3′ regulatory region of MIR160a. foc plants exhibit a variety of intriguing phenotypes, including serrated rosette leaves, irregular flowers, floral organs inside siliques, reduced fertility, aberrant seeds, and viviparous seedlings. Detailed phenotypic analysis showed that abnormal cell divisions in the basal embryo domain and suspensor led to diverse defects during embryogenesis in foc plants. Further analysis showed that the 3′ region was required for the expression of MIR160a. The accumulation of mature miR160 was greatly reduced in foc inflorescences. In addition, the expression pattern of ARF16 and -17 was altered during embryo development in foc plants. foc plants were also deficient in auxin responses. Moreover, auxin was involved in regulating the expression of MIR160a through its 3′ regulatory region. Our study not only provides insight into the molecular mechanism of embryo development via MIR160a-regulated ARFs, but also reveals the mechanism regulating MIR160a expression.
Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small ...protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.
Scanning Electron Microscope (SEM) as one of the major research and industrial equipment for imaging of micro-scale samples and surfaces has gained extensive attention from its emerge. However, the ...acquired micrographs still remain two-dimensional (2D). In the current work a novel and highly accurate approach is proposed to recover the hidden third-dimension by use of multi-view image acquisition of the microscopic samples combined with pre/post-processing steps including sparse feature-based stereo rectification, nonlocal-based optical flow estimation for dense matching and finally depth estimation. Employing the proposed approach, three-dimensional (3D) reconstructions of highly complex microscopic samples were achieved to facilitate the interpretation of topology and geometry of surface/shape attributes of the samples. As a byproduct of the proposed approach, high-definition 3D printed models of the samples can be generated as a tangible means of physical understanding. Extensive comparisons with the state-of-the-art reveal the strength and superiority of the proposed method in uncovering the details of the highly complex microscopic samples.
Recent advances in 3D SEM surface reconstruction Tafti, Ahmad P.; Kirkpatrick, Andrew B.; Alavi, Zahrasadat ...
Micron (Oxford, England : 1993),
November 2015, 2015-Nov, 2015-11-00, 20151101, Letnik:
78
Journal Article
Recenzirano
•An attempt is made to explain the expanding body of 3D SEM surface reconstruction algorithms.•We develop an optimized multi-view framework for the 3D SEM surface reconstruction problem.•We analyze ...the qualitative and quantitative attributes of our proposed framework from different perspectives. The results are promising.•We model a taxonomy of 3D SEM surface reconstruction approaches and address several challenging issues as part of our future work.•This contribution provides insights and tendencies, and plans several future enhancements to advance the level of 3D surface reconstruction for SEM images.
The scanning electron microscope (SEM), as one of the most commonly used instruments in biology and material sciences, employs electrons instead of light to determine the surface properties of specimens. However, the SEM micrographs still remain 2D images. To effectively measure and visualize the surface attributes, we need to restore the 3D shape model from the SEM images. 3D surface reconstruction is a longstanding topic in microscopy vision as it offers quantitative and visual information for a variety of applications consisting medicine, pharmacology, chemistry, and mechanics. In this paper, we attempt to explain the expanding body of the work in this area, including a discussion of recent techniques and algorithms. With the present work, we also enhance the reliability, accuracy, and speed of 3D SEM surface reconstruction by designing and developing an optimized multi-view framework. We then consider several real-world experiments as well as synthetic data to examine the qualitative and quantitative attributes of our proposed framework. Furthermore, we present a taxonomy of 3D SEM surface reconstruction approaches and address several challenging issues as part of our future work.
Purpose In addition to prospective trials for non-small-cell lung cancers (NSCLCs) that are driven by less common genomic alterations, registries provide complementary information on patient response ...to targeted therapies. Here, we present the results of an international registry of patients with RET-rearranged NSCLCs, providing the largest data set, to our knowledge, on outcomes of RET-directed therapy thus far. Methods A global, multicenter network of thoracic oncologists identified patients with pathologically confirmed NSCLC that harbored a RET rearrangement. Molecular profiling was performed locally by reverse transcriptase polymerase chain reaction, fluorescence in situ hybridization, or next-generation sequencing. Anonymized data-clinical, pathologic, and molecular features-were collected centrally and analyzed by an independent statistician. Best response to RET tyrosine kinase inhibition administered outside of a clinical trial was determined by RECIST v1.1. Results By April 2016, 165 patients with RET-rearranged NSCLC from 29 centers across Europe, Asia, and the United States were accrued. Median age was 61 years (range, 29 to 89 years). The majority of patients were never smokers (63%) with lung adenocarcinomas (98%) and advanced disease (91%). The most frequent rearrangement was KIF5B-RET (72%). Of those patients, 53 received one or more RET tyrosine kinase inhibitors in sequence: cabozantinib (21 patients), vandetanib (11 patients), sunitinib (10 patients), sorafenib (two patients), alectinib (two patients), lenvatinib (two patients), nintedanib (two patients), ponatinib (two patients), and regorafenib (one patient). The rate of any complete or partial response to cabozantinib, vandetanib, and sunitinib was 37%, 18%, and 22%, respectively. Further responses were observed with lenvantinib and nintedanib. Median progression-free survival was 2.3 months (95% CI, 1.6 to 5.0 months), and median overall survival was 6.8 months (95% CI, 3.9 to 14.3 months). Conclusion Available multikinase inhibitors had limited activity in patients with RET-rearranged NSCLC in this retrospective study. Further investigation of the biology of RET-rearranged lung cancers and identification of new targeted therapeutics will be required to improve outcomes for these patients.
Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis ...(Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.
COVID-19 restrictions led to reduced levels of physical activity, increased screen usage, and declines in mental health in youth; however, in-depth understandings of the experiences of high school ...student-athletes have yet to be explored. To describe the experiences of the COVID-19 pandemic on student-athletes' physical activity, social connection, and mental health, 20 high school student-athletes living in Calgary, Alberta participated in semi-structured interviews, designed using phenomenography. Participants reported variations in physical activity, social connections, and mental health which were influenced by stay-at-home restrictions and weather. Access to resources, changes to routines, online classes, and social support all influenced engagement in physical activity. School and sports provided opportunities for in-person social connections, impacted by the onset of the pandemic. Participants reported their mental health was influenced by social connections, online classes, and physical activity. Findings from this study will inform the development of resources for high school student-athletes amidst COVID-19.
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