Search

Commonly referred to as the maternal-to-zygotic transition, the shift of developmental control from maternal-to-zygotic genomes is a key event during animal and plant embryogenesis. Together with the degradation of parental gene products, the increased transcriptional activities of the zygotic genome remodels the early embryonic transcriptome during this transition. Although evidence from multiple flowering plants suggests that zygotes become transcriptionally active soon after fertilization, the timing and developmental requirements of zygotic genome activation in Arabidopsis thaliana (Arabidopsis) remained a matter of debate until recently. In this report, we optimized an expansion microscopy technique for robust immunostaining of Arabidopsis ovules and seeds. This enabled the detection of marks indicative of active transcription in zygotes before the first cell division. Moreover, we employed a live-imaging culture system together with transcriptional inhibitors to demonstrate that such active transcription is physiologically required in zygotes and early embryos. Our results indicate that zygotic genome activation occurs soon after fertilization and is required for the initial zygotic divisions in Arabidopsis.

The application of single-cell mRNA sequencing technologies to plant embryos promises to reveal the gene expression dynamics underlying cell-type differentiation. A new paper in Development reports the generation of high-quality transcriptomes from single embryonic nuclei without contamination from maternal tissues. To find out more about the story, we caught up with first author Ping Kao and his supervisor Michael Nodine, who recently moved from the Gregor Mendel Institute in Vienna to become Assistant Professor in the Laboratory of Molecular Biology at Wageningen University in the Netherlands.

In the high-speed differential (HSD) interface design, the intra-pair skew becomes more critical as the link speed is ever increasing beyond 56Gbps+ signaling. The skew within a differential pair (intra-pair skew) deteriorates eye opening at the receiver and degrades the channel performance significantly in HSD signaling. However, industry lacks in an accurate and stable intra-pair skew measurement methodology, which created a significant gap in interconnect testing and qualification. In this paper, the effective intra-pair skew (EIPS) is introduced, which is directly calculated from S-parameter data as a single value without time-domain conversion. The EIPS is verified by correlating to the eye opening and the common-mode noise (CMN) in the HSD link of PCIe Gen5 32Gbps NRZ signaling. The MATLAB script for the EIPS calculation is included in the appendix.

Expansion microscopy (ExM) improves image resolution of specimens without requirements of sophisticated techniques or equipment. Probes or proteins are anchored onto an acrylamide gel matrix which is then expanded with osmotic pressure. As the physical distance between two signal points increases, previously confounded signals can be resolved while their relative spatial locations are retained. ExM has been successfully applied to several animal tissues, but its application to plant tissues was only recently demonstrated. Here we provide a detailed ExM protocol for plant tissues using fluorescent immunostaining of developing Arabidopsis thaliana (Arabidopsis) seeds as an example. This modified ExM protocol enables expansion of ovule/seed samples, and preserves the majority of fluorescent protein signals in the expanded samples. The fluorescent immunostaining observed using this protocol demonstrates the feasibility of detecting cellular events and subcellular structures in expanded plant samples. This ExM protocol variant for plants can serve as a guideline for applying ExM to various plant tissues and help increase the resolution of corresponding microscopy based studies.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated ‘The people behind the papers’ interview., Highlighted Article: A transcriptome atlas of Arabidopsis embryos constructed from single nuclei reveals cell-specific epigenetic and transcriptional regulatory features.

Expansion microscopy (ExM) improves image resolution of specimens without requirements of sophisticated techniques or equipment. Probes or proteins are anchored onto an acrylamide gel matrix which is then expanded with osmotic pressure. As the physical distance between two signal points increases, previously confounded signals can be resolved while their relative spatial locations are retained. ExM has been successfully applied to several animal tissues, but its application to plant tissues was only recently demonstrated. Here we provide a detailed ExM protocol for plant tissues using fluorescent immunostaining of developing Arabidopsis thaliana (Arabidopsis) seeds as an example. This modified ExM protocol enables expansion of ovule/seed samples, and preserves the majority of fluorescent protein signals in the expanded samples. The fluorescent immunostaining observed using this protocol demonstrates the feasibility of detecting cellular events and subcellular structures in expanded plant samples. This ExM protocol variant for plants can serve as a guideline for applying ExM to various plant tissues and help increase the resolution of corresponding microscopy based studies.

Eukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development, and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation in eukaryotes, but little is known about how chromatin regulates siRNA transcription. We demonstrated that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. Depending on whether they are located in euchromatic or heterochromatic regions of the genome, bipartite-classified TEs generate siRNAs throughout embryogenesis according to two distinct patterns. siRNAs are transcribed in embryos and required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states. Decondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.

Cogon grass (Imperata cylindrica (L.) Beauv. var. major (Nees) Hubb.) is one of the top-ten weeds worldwide. It is also a C4 medicinal plant. In particular, an ecotype from Chuwei (CW) mangrove forest was found to be salt tolerant. Comparative proteomic analysis using two-dimensional (2D)-difference in gel electrophoresis coupled with liquid chromatography-mass spectrometry (LC-MS) was carried out to identify responsive leaf proteins in the CW ecotype and salt-intolerant Sarlun (SL) population following three days of 150 mM sodium chloride salt stress treatment. We identified five photosynthesis proteins including Rubisco small subunit, uncharacterized protein LOC100194054, Cyt b6-f, oxygen-evolving enhancer 2, and photosystem I reaction center subunit IV which were significantly up- or down-regulated by salt stress in CW ecotype but not SL population. Gene ontology enrichment analysis showed that photosynthesis was over-represented. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008482. Taken together, our proteomic study identified differentially accumulated proteins which provide additional evidence of ecophysiological variation in two natural variants of I. cylindrica.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.

Genome-wide characterization of RNA populations in early flowering plant embryos can yield insights into the gene regulatory processes functioning during this formative phase of development. However, early embryonic transcriptomes are technically challenging to profile because of the low amount of RNA obtainable and potential RNA contamination from surrounding nonembryonic tissues. Here we provide a detailed protocol for collecting early Arabidopsis thaliana (Arabidopsis) embryos, generating mRNA sequencing (mRNA-seq) libraries, and basic data processing and quality controls of the resulting mRNA-seq data.

Genome-wide characterization of RNA populations in early flowering plant embryos can yield insights into the gene regulatory processes functioning during this formative phase of development. However, early embryonic transcriptomes are technically challenging to profile because of the low amount of RNA obtainable and potential RNA contamination from surrounding nonembryonic tissues. Here we provide a detailed protocol for collecting early Arabidopsis thaliana (Arabidopsis) embryos, generating mRNA sequencing (mRNA-seq) libraries, and basic data processing and quality controls of the resulting mRNA-seq data.

Demographic structure of Taiwan is becoming an aging society with fewer children due to lower birth rate, which have great impacts on the teacher supply and demand, education resources and the school education. It is a currently important issue to concern the future education developments from the standpoint of demographic transition. In recent years, because of socio-economic pressures and social values change, the birth population of Taiwan is dramatically decreased, the birth rate declines, and the society of the trend of fewer children is formed. The crisis and the impact produced by the trend of fewer children directly reflect on educations. We collect date related to the source of students, including whole populations of Taiwan, birth population of Taiwan, the ratio of class number to student number from all high schools in Taiwan, enrollment number, enrollment grades and ratios of vocational schools in Taiwan, to predict the situation of the future source of students in variety collage departments based on the collecting data. By surveying the ninth grade student and the high school third grade student as subjects, their choosing intention of further studies is validated, then the ratio of ambitions and the school ranks is adjusted. In this study “Analysis model of college student sources”, we used the Visual Basic and C# language to write a college student source inquiring website through which the trend of application number and the birth population of the school year are obtained. By this study, we can estimate the situation of the possible low enrollment, and provide it as a consult to schools for recruiting new students or for transformation, meanwhile this study provides sufficient data analysis as a reference basis for students when deciding the school, to prevent from choosing closing departments or schools, and provide application-related information to user as the reference.

Interfering oncogenic STAT3 signaling is a promising anti-cancer strategy. We examined the efficacy and drug mechanism of an obatoclax analog SC-2001, a novel STAT3 inhibitor, in human breast cancer cells. Human breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and western blot. Signaling pathways were assessed by western blot. In vivo efficacy of SC-2001 was tested in xenograft nude mice. SC-2001 inhibited cell growth and induced apoptosis in association with downregulation of p-STAT3 (Tyr 705) in breast cancer cells. STAT3-regulated proteins, including Mcl-1, survivin, and cyclin D1, were repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of protein tyrosine phosphatase SHP-1, a negative regulator of STAT3. Furthermore, the enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription by RFX-1. Chromatin immunoprecipitation assay revealed that SC-2001 increased the binding capacity of RFX-1 to the SHP-1 promoter. Knockdown of either RFX-1 or SHP-1 reduced SC-2001-induced apoptosis, whereas ectopic expression of RFX-1 increased SHP-1 expression and enhanced the apoptotic effect of SC-2001. Importantly, SC-2001 suppressed tumor growth in association with enhanced RFX-1 and SHP-1 expression and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. SC-2001 induced apoptosis in breast cancer cells, an effect that was mediated by RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. Our study indicates targeting STAT3 signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer.

Self-assembled monolayers (SAMs) of nitrile-substituted oligo(phenylene ethynylene) thiols (NC-OPEn) with a variable chain length n (n ranging from one to three structural units) on Au(111) were studied by synchrotron-based high-resolution X-ray photoelectron spectroscopy and near-edge absorption fine-structure spectroscopy. The experimental data suggest that the NC-OPEn molecules form well-defined SAMs on Au(111), with all the molecules bound to the substrate through the gold–thiolate anchor and the nitrile tail groups located at the SAM–ambient interface. The packing density in these SAMs was found to be close to that of alkanethiolate monolayers on Au(111), independent of the chain length. Similar behavior was found for the molecular inclination, with an average tilt angle of ~33–36° for all the target systems. In contrast, the average twist of the OPEn backbone (planar conformation) was found to depend on the molecular length, being close to 45° for the films comprising the short OPE chains and ~53.5° for the long chains. Analysis of the data suggests that the attachment of the nitrile moiety, which served as a spectroscopic marker group, to the OPEn backbone did not significantly affect the molecular orientation in the SAMs.

In this paper, we use micromachined, high-frequency, quartz bulk acoustic wave resonators to systematically study the physical and viscoelastic properties of spontaneously adsorbed globular protein films with molecular weights (MWs) spanning three decades on hydrophobic surfaces. Specifically, changes in the frequency and the Q -factor of the micromachined resonator array were studied as a function of concentration for five proteins, namely human serum albumin (HSA), immunoglobulin G (IgG), human fibrinogen (Fib), alpha-2-macroglobulin (AMG), and immunoglobulin M (IgM) at the fundamental and third resonance modes. The results obtained were interpreted using equivalent electrical impedance models for the multilayer stack on the quartz crystal microbalances surface. Discrete changes in the protein adsorption and the viscoelastic behavior with solution concentration were observed for all the five protein films. The spherical core-shell protein model is used to provide a simple explanation of the results. The work presents the first systematic and quantitative evaluation of the density, thickness, viscosity, and elastic modulus of adsorbed globular protein films and demonstrates the advantages of using micromachined high-frequency bulk acoustic wave resonators for obtaining these types of data.

This paper presents the design, fabrication, and characterization of thermal infrared (IR) imaging arrays operating at room temperature which are based on Y-cut-quartz bulk acoustic wave resonators. A novel method of tracking the resonance frequency based upon the measurement of impedance is presented. High-frequency (240-MHz) micromachined resonators from Y-cut-quartz crystal cuts were fabricated using heterogeneous integration techniques on a silicon wafer. A temperature sensitivity of 22.16 kHz/°C was experimentally measured. IR measurements on the resonator pixel resulted in a noise equivalent power of 3.90 nW/Hz1/2, a detectivity D* of 1 × 105 cm · Hz1/2/W, and a noise equivalent temperature difference of 4 mK in the 8- to 14-μm wavelength range. The thermal frequency response of the resonator was determined to be faster than 33 Hz, demonstrating its applicability in video-rate uncooled IR imaging. This work represents the first comprehensive thermal characterization of micromachined F-cut-quartz resonators and their IR sensing response.

The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square-or-hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square-or-hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM.