miR160 and miR166/165 Contribute to the LEC2-Mediated Auxin Response Involved in the Somatic Embryogenesis Induction in Arabidopsis

MicroRNAs are non-coding small RNA molecules that are involved in the post-transcriptional regulation of the genes that control various developmental processes in plants, including zygotic embryogenesis (ZE). miRNAs are also believed to regulate somatic embryogenesis (SE), a counterpart of the ZE that is induced in vitro in plant somatic cells. However, the roles of specific miRNAs in the regulation of the genes involved in SE, in particular those encoding transcription factors (TFs) with an essential function during SE including LEAFY COTYLEDON2 (LEC2), remain mostly unknown. The aim of the study was to reveal the function of miR165/166 and miR160 in the LEC2-controlled pathway of SE that is induced in in vitro cultured Arabidopsis explants.In ZE, miR165/166 controls the PHABULOSA/PHAVOLUTA (PHB/PHV) genes, which are the positive regulators of LEC2, while miR160 targets the AUXIN RESPONSE FACTORS (ARF10, ARF16, ARF17) that control the auxin signaling pathway, which plays key role in LEC2-mediated SE. We found that a deregulated expression/function of miR165/166 and miR160 resulted in a significant accumulation of auxin in the cultured explants and the spontaneous formation of somatic embryos. Our results show that miR165/166 might contribute to SE induction via targeting PHB, a positive regulator of LEC2 that controls embryogenic induction via activation of auxin biosynthesis pathway (Wójcikowska et al., 2013). Similar to miR165/166, miR160 was indicated to control SE induction through auxin-related pathways and the negative impact of miR160 on ARF10/ARF16/ARF17 was shown in an embryogenic culture. Altogether, the results suggest that the miR165/166- and miR160-node contribute to the LEC2-mediated auxin-related pathway of embryogenic transition that is induced in the somatic cells of Arabidopsis. A model summarizing the suggested regulatory interactions between the miR165/166-PHB and miR160-ARF10/ARF16/ARF17 nodes that control SE induction in Arabidopsis was proposed

Whole Mount in situ Localization of miRNAs and mRNAs During Somatic Embryogenesis in Arabidopsis

Somatic embryogenesis (SE) results from the transition of differentiated plant somatic cells into embryogenic cells that requires the extensive reprogramming of the somatic cell transcriptome. Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount in situ hybridization (WISH) method (Bleckmann and Dresselhaus, 2016; Dastidar et al., 2016) in order to analyze the spatiotemporal localization of miRNAs (miR156, miR166, miR390, miR167) and mRNAs such as WOX5 and PHABULOSA-target of miR165/166 during the SE that is induced in Arabidopsis explants. This study presents a detailed step-by-step description of the WISH procedure in which DIG-labeled LNA and RNA probes were used to detect miRNAs and mRNAs, respectively. The usefulness of the WISH in the functional analysis of the SE-involved regulatory pathways is demonstrated and the advantages of this method are highlighted: (i) the ability to analyze intact non-sectioned plant tissue; (ii) the specificity of transcript detection; (iii) the detection of miRNA; and (iv) a semi-quantitative assessment of the RNA abundance

Detecting the “gist” of breast cancer in mammograms three years before localized signs of cancer are visible

Objectives: After a 500 ms presentation, experts can distinguish abnormal mammograms at above chance levels even when only the breast contralateral to the lesion is shown. Here, we show that this signal of abnormality is detectable 3 years before localized signs of cancer become visible. Methods: In 4 prospective studies, 59 expert observers from 3 groups viewed 116–200 bilateral mammograms for 500 ms each. Half of the images were prior exams acquired 3 years prior to onset of visible, actionable cancer and half were normal. Exp. 1D included cases having visible abnormalities. Observers rated likelihood of abnormality on a 0–100 scale and categorized breast density. Performance was measured using receiver operating characteristic analysis. Results: In all three groups, observers could detect abnormal images at above chance levels 3 years prior to visible signs of breast cancer (p < 0.001). The results were not due to specific salient cases nor to breast density. Performance was correlated with expertise quantified by the number of mammographic cases read within a year. In Exp. 1D, with cases having visible actionable pathology included, the full group of readers failed to reliably detect abnormal priors; with the exception of a subgroup of the six most experienced observers. Conclusions: Imaging specialists can detect signals of abnormality in mammograms acquired years before lesions become visible. Detection may depend on expertise acquired by reading large numbers of cases. Advances in knowledge: Global gist signal can serve as imaging risk factor with the potential to identify patients with elevated risk for developing cancer, resulting in improved early cancer diagnosis rates and improved prognosis for females with breast cancer

A Continuous Automated Vault Inventory System (CAVIS) for accountability monitoring of stored nuclear materials

Nearly all facilities that store hazardous (radioactive or non-radioactive) materials must comply with prevailing federal, state, and local laws. These laws usually have components that require periodic physical inspections to insure that all materials remain safely and securely stored. The inspections are generally labor intensive, slow, put personnel at risk, and only find anomalies after they have occurred. The system described in this paper was developed for monitoring stored nuclear materials resulting from weapons dismantlement, but its applications extend to any storage facility that meets the above criteria. The traditional special nuclear material (SNM) accountability programs, that are currently used within most of the Department of Energy (DOE) complex, require the physical entry of highly trained personnel into SNM storage vaults. This imposes the need for additional security measures, which typically mandate that extra security personnel be present while SNM inventories are performed. These requirements increase labor costs and put additional personnel at risk to radiation exposure. In some cases, individuals have received radiation exposure equivalent to the annual maximum during just one inventory verification. With increasing overhead costs, the current system is rapidly becoming too expensive to operate, the need for an automated method of inventory verification is evident. The Continuous Automated Vault Inventory System (CAVIS) described in this paper was designed and prototyped as a low cost, highly reliable, and user friendly system that is capable of providing, real-time weight, gamma. and neutron energy confirmation from each item stored in a SNM vault. This paper describes the sensor technologies, the CAVIS prototype system (built at Y- 12 for highly enriched uranium storage), the technical requirements that must be achieved to assure successful implementation, and descriptions of sensor technologies needed for a plutonium facility

Identification of genes preferentially expressed in wheat egg cells and zygotes

Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat

A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms

The extent and variability of storm-induced temperature changes in lakes measured with long-term and high-frequency data

The intensity and frequency of storms are projected to increase in many regions of the world because of climate change. Storms can alter environmental conditions in many ecosystems. In lakes and reservoirs, storms can reduce epilimnetic temperatures from wind-induced mixing with colder hypolimnetic waters, direct precipitation to the lake's surface, and watershed runoff. We analyzed 18 long-term and high-frequency lake datasets from 11 countries to assess the magnitude of wind- vs. rainstorm-induced changes in epilimnetic temperature. We found small day-to-day epilimnetic temperature decreases in response to strong wind and heavy rain during stratified conditions. Day-to-day epilimnetic temperature decreased, on average, by 0.28 degrees C during the strongest windstorms (storm mean daily wind speed among lakes: 6.7 +/- 2.7 m s(-1), 1 SD) and by 0.15 degrees C after the heaviest rainstorms (storm mean daily rainfall: 21.3 +/- 9.0 mm). The largest decreases in epilimnetic temperature were observed >= 2 d after sustained strong wind or heavy rain (top 5(th) percentile of wind and rain events for each lake) in shallow and medium-depth lakes. The smallest decreases occurred in deep lakes. Epilimnetic temperature change from windstorms, but not rainstorms, was negatively correlated with maximum lake depth. However, even the largest storm-induced mean epilimnetic temperature decreases were typicallyPeer reviewe