Fast virtual histology using X-ray in-line phase tomography: application to the 3D anatomy of maize developing seeds

Background

Despite increasing demand, imaging the internal structure of plant organs or tissues without the use of transgenic lines expressing fluorescent proteins remains a challenge. Techniques such as magnetic resonance imaging, optical projection tomography or X-ray absorption tomography have been used with various success, depending on the size and physical properties of the biological material.

Results

X-ray in-line phase tomography was applied for the imaging of internal structures of maize seeds at early stages of development, when the cells are metabolically fully active and water is the main cell content. This 3D imaging technique with histology-like spatial resolution is demonstrated to reveal the anatomy of seed compartments with unequalled contrast by comparison with X-ray absorption tomography. An associated image processing pipeline allowed to quantitatively segment in 3D the four compartments of the seed (embryo, endosperm, nucellus and pericarp) from 7 to 21 days after pollination.

Conclusion

This work constitutes an innovative quantitative use of X-ray in-line phase tomography as a non-destructive fast method to perform virtual histology and extends the developmental stages accessible by this technique which had previously been applied in seed biology to more mature samples.

     

A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM To explore the molecular mechanism of transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） and cisplatin resistance in three-dimensionally cultured lung cancer cells. METHODS Under three-dimensional culture condition, the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy, scanning electron microscopy, laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation. The cisplatin sensitivity was determined by MTT assay. RESULTS Under the three-dimensional culture condition, the structure of 95D cell spheroids after TGF-β1 stimulation collapsed, the cells were dispersed and migrating, and the spheroids merged with each other. The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation, and the protein expression of N-cadherin and vimentin was significantly up-regulated. The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation, and the protein levels of N-cadherin, vimentin, phosphorylated AKT and phosphorylated mTOR were up-regulated. LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins. The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin, while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1. CONCLUSION TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.     

山杏下胚轴再生不定芽影响因子研究(英文)

山杏成熟种子在改良MS培养基上培养获得下胚轴,将其切段后培养在改良MS培养基+TDZ2.0mg/L+NAA0.5mg/L的再生培养基上,经15d的暗期培养后,其再生频率可达到37%以上。试验表明分裂素TDZ促进不定芽再生的效果优于6-BA,生长素NAA效果优于2,4-D,2-3周龄的下胚轴再生效果较好,但下胚轴的取材部位对再生无显著影响。     

A novel,repeatable, and non-destructive method to collect root xylem exudate from mature apple trees in an orchard and to compare its constituents with shoot exudate

SUMMARY

A method is described in detail to allow repeatable and non-destructive extraction of xylem exudate from the trunk of mature apple trees (Malus domestica Borkh.) by the use of reduced air pressure. The method allows extraction of sufficient uncontaminated exudate to determine and quantify constituents such as plant hormones, sugars, amino acids, and minerals. Analytical measurements from the first experimental year were conducted to determine the minimum volume of exudate required to obtain reliable values for various exudate constituents. Some constituents of root exudates differed compared to shoot exudates obtained by traditional destructive methods. Thus, shoot exudates may be less suitable if the influence of roots on the aerial parts of a tree is being studied (e.g., in relation to environmental conditions or horticultural practices such as root pruning, girdling, irrigation or fertilisation). The extraction method worked well between Spring and mid-Summer over 3 years. However, the procedure failed from the end of June, onwards. Possible reasons for this failure are discussed, and experiments which may improve lateseason sap recovery are suggested. Small modifications to the method should allow simultaneous, non-destructive extraction of root and shoot-exudates, permitting a more coherent picture of long-distance signals from root-to-shoot, and vice versa, and their co-ordinating significance.     

A computer-based tool to link decay information to 3D architecture of urban trees

We present a software application suitable for studies of the decay in trees. The application implements the storage of measurements of stem characteristics (related to decay) and visualization of the three-dimensional (3D) structural model of tree stems and branches built using this material. The application has been constructed on the basis of an existing architectural model of trees. A set of photographs taken from discs cut from felled trees works as the input for the application. The user will define, using the application, the contours of the areas of interest (e.g. healthy wood, decayed wood and cavity) manually in the photographs of stem or branch cross sections. The application then builds a 3D structural model of stem and/or branches on the basis of the input. It is possible to study visually the composition of the stem and/or branches of different types of wood. This helps to evaluate the location of potential hazard and its relation to existing damage in tree stems and branches.     

A method to calculate the number of wheat seedlings in the 1st to the 3rd leaf growth stages

Background

The number of cultivated wheat seedlings per unit area allows calculation of plant density. Wheat seedling density provides emergence data and this is useful for improving crop management. The number of wheat seedlings is typically determined by visual counts but this is time-consuming and laborious.

Results

We obtained field digital images of 1st to 3rd leaf stage wheat seedlings. The seedlings were extracted using an image analysis technique that calculated the coverage degree of the seedlings and the number of angular points of overlapping leaves. The wheat seedling quantity estimation model was constructed using multivariate regression analysis. The model parameters included coverage degree, number of angular points, variety coefficient, and leaf age. Introduction of the number of angular points increased the accuracy of the single coverage degree model. The R² value was consistently?>?0.95 when the model was applied to different varieties, indicating that the model was adaptable for different varieties. As the leaf stage or density increased, the accuracy of the model declined, but the minimum R² remained?>?0.87, indicating good adaptability of the model to seedlings with different leaf ages and densities.

Conclusions

This method is an effective means for counting wheat seedlings in the 1st to the 3rd leaf stages.

     

Segmentation of 3D images of plant tissues at multiple scales using the level set method

Background

Developmental biology has made great strides in recent years towards the quantification of cellular properties during development. This requires tissues to be imaged and segmented to generate computerised versions that can be easily analysed. In this context, one of the principal technical challenges remains the faithful detection of cellular contours, principally due to variations in image intensity throughout the tissue. Watershed segmentation methods are especially vulnerable to these variations, generating multiple errors due notably to the incorrect detection of the outer surface of the tissue.

Results

We use the level set method (LSM) to improve the accuracy of the watershed segmentation in different ways. First, we detect the outer surface of the tissue, reducing the impact of low and variable contrast at the surface during imaging. Second, we demonstrate a new edge function for a level set, based on second order derivatives of the image, to segment individual cells. Finally, we also show that the LSM can be used to segment nuclei within the tissue.

Conclusion

The watershed segmentation of the outer cell layer is demonstrably improved when coupled with the LSM-based surface detection step. The tool can also be used to improve watershed segmentation at cell-scale, as well as to segment nuclei within a tissue. The improved segmentation increases the quality of analysis, and the surface detected by our algorithm may be used to calculate local curvature or adapted for other uses, such as mathematical simulations.

     

A simple and efficient method for the long-term preservation of plant cell suspension cultures

Background  

The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.     

Evaluating the effect of 3D urban form on neighborhood land surface temperature using Google Street View and geographically weighted regression

Landscape Ecology - Land surface temperature (LST) directly responds to incoming solar radiation and is strongly influenced by vertical urban structures, such as trees and buildings that provide...     

NIH3T3 cell proliferation and cell cycle in the conditions of hypoxia or low nutrition and its response to bFGF

AIM: To investigate NIH3T3 cell proliferation and cell cycle in the condition of hypoxia or low nutrition and its response to bFGF, and to explore the pathophysiological changes of fibroblast under hypoxic or low nutrional conditions.METHODS: The cells were placed in anaerobic workstation where the mixture gas was given to simulate hypoxic environment. Partial oxygen pressure (PO2) of medium was controlled in 27 mmHg, 44 mmHg and 175mmHg. NIH3T3 cells were cultured with low nutritional medium contained new bovine serum (NCS) less than 10% to simulate low nutritional environment. MTT assay was used for observing cell activity and flow cytometry for cell cycle analysis.RESULTS: Under 44 mmHg PO2, no obvious difference was shown between hypoxia group and normal group. Under 27 mmHg PO2, the proliferation activity of NIH3T3 cells was significantly lower than that in normal group (P<0.01), as well as the cell numbers in G0-G1 phase increased (P<0.05), S phase decreased (P<0.01). bFGF had no effect on cell proliferation. In 0.5% NCS medium, the NIH3T3 cell proliferation speed decreased (P<0.01) and cell cycle was arrested at G0-G1 (P<0.01). The proliferation speed was improved by bFGF (P<0.01).CONCLUSION: In lower PO2 or lower nutrinal condition, fibroblast proliferation activity is inhibited by cell cycle arrest in G0/G1 phase. However the decreasing proliferation in low nutritional medium could be improved by external bFGF.     

A fuzzy logic method to assess the relationship between landscape patterns and bird richness of the Rolling Pampas

The loss of biodiversity in productive ecosystems is a global concern of the last decades. The Rolling Pampas of Argentina is an intensively cropped region that underwent important land use and landscape change, with different impacts on biodiversity of both plants and animals. Land use type and habitat complexity are hypothesized to be the most important factors determining species richness in agro-ecosystems. But it is not easy to define these attributes in an unambiguous fashion, or determine their interactions at different spatial scales. A fuzzy logic approach allows overcoming some of these problems by using linguistic variables and logic rules to relate them and formulate hypothesis. We constructed fuzzy logic models to study how bird species richness in the Rolling Pampas is related to land use and habitat complexity, and how these variables interact at two spatial scales. Results showed that at the local scale, landscape complexity is the most important factor determining species numbers; trees and bodies of water are the most influential complexities. The effect of local scale landscape attributes was modified depending on the context at broader scales, so that agricultural sites were enriched when surrounded by more favorable landscapes. There was a high dispersion in the predicted/observed value relationship, indicating that landscape factors interact in more complex ways than those captured by the models we used. We suggest that the fuzzy logic approach is suitable for working with biological systems, and we discuss the advantages and disadvantages of its use.     

A spatially-explicit method to assess the dry deposition of air pollution by urban forests in the city of Florence,Italy

Urban forests (UF) provide a range of important ecosystem services (ES) for human well-being. Relevant ES delivered by UF include urban temperature regulation, runoff mitigation, noise reduction, recreation, and air purification. In this study the potential of air pollution removal by UF in the city of Florence (Italy) was investigated. Two main air pollutants were considered – particulate matter (PM₁₀) and tropospheric ozone (O₃) – with the aim of providing a methodological framework for mapping air pollutant removal by UF and assessing the percent removal of air pollutant.The distribution of UF was mapped by high spatial resolution remote sensing data and classified into seven forest categories. The Leaf Area Index (LAI) was estimated spatially using a regression model between in-field LAI survey and Airborne Laser Scanning data and it was found to be in good linear agreement with estimates from ground-based measurements (R² = 0.88 and RMSE% = 11%). We applied pollution deposition equations by using pollution concentrations measured at urban monitoring stations and then estimated the pollutant removal potential of the UF: annual O₃ and PM₁₀ removal accounted for 77.9 t and 171.3 t, respectively. O₃ and PM₁₀ removal rates by evergreen broadleaves (16.1 and 27.3 g/m²), conifers (10.9 and 28.5 g/m²), and mixed evergreen species (15.8 and 31.7 g/m²) were higher than by deciduous broadleaf stands (4.1 and 10 g/m²). However, deciduous forests exhibited the largest total removal due to the high percentage of tree cover within the city. The present study confirms that UF play an important role in air purification in Mediterranean cities as they can remove monthly up to 5% of O₃ and 13% of PM₁₀.     

甜樱桃砧木PcMPK3基因启动子的克隆及对病原菌感染的响应

【目的】探明PcMPK3基因的表达调控规律。【方法】利用染色体步移技术从甜樱桃矮化砧木Gisela 6中克隆PcMPK3基因的启动子序列PcMPK3pro。利用Neural Network Promotor Prediction、softberry、PLACE和PlantCARE网站在线预测PcMPK3基因的基础启动子、转录起始位点和顺式作用元件。将PcMPK3pro定向替换植物表达载体pBI121-SN1的CaMV35S组成型启动子,构建重组表达载体pBI-PcMPK3pro:GUS,瞬时转化烟草叶片。【结果】结果表明,PcMPK3pro含有启动子核心元件TATA-box和CAAT-box等多种响应胁迫的顺式作用元件。受病原菌丁香假单胞菌番茄致病变种(Pseudomonas syringae pv. tomato DC3000,Pst DC3000)侵染,PcMPK3pro能驱动GUS报告基因表达且GUS酶活性显著提高。【结论】推测PcMPK3基因参与植物响应病原菌感染的胁迫过程。     

Biomonitoring of ozone: A tool to initiate the young people into the scientific method and environmental issues. A case study in Central Italy

The wide diffusion of tropospheric ozone is a major environmental problem, in urban area as well as in rural and remote localities. Its increasing man-related levels are connected to severe impacts on human life and welfare, in terms of adverse health effects, damage to manufacts and injury to plants. Biological monitoring is a powerful tool for filling the gap between the causes and the effects of environmental toxins, as bioindication agents assess in an easy-to-detect fashion the effects of pollution on (selected) biota. A project was launched in 2012 to involve some 190 students (ages 11–16) from three schools in Central Italy in biodetection of ozone effects with the hypersensitive plant Nicotiana tabacum Bel-W3. The project also involved teachers and families of the pupils. Results implied the reading of 12,000 biological data (ozone injury on cotyledons) and were fortified by data captured by four automatic analyzers (1300 raw data of hourly means). Biological and chemical data compared favorably and were treated with geostatistical methods; results are exposed in the form of cartographic restitutions. Under the guidance of their teachers, the pupils had several opportunities to practice with many basic and applied study areas and disciplines and were initiated into the scientific method in a simple and absorbing manner. Though primarily an educational exercise, the survey provided sound research elements and the picture of pollution that emerged has increased the knowledge of air quality in the area. Biomonitoring is confirmed to be a powerful tool to involve young people in environmental topics.     

lncRNA TTN-AS1 regulates the viability and invasion of lung adenocarcinoma A549 cell via miR-519d-3p/MMP2 pathway

AIM To investigate the expression level of long noncoding RNA （lncRNA） TTN antisense RNA 1 （TTN-AS1） in lung adenocarcinoma tissues and the effects of TTN-AS1 silencing on the viability and invasion of lung adenocarcinoma A549 cells. METHODS RT-qPCR was used to detect the expression of TTN-AS1, microRNA-519d-3p （miR-519d-3p） and matrix metalloproteinase 2 （MMP2） mRNA in 32 cases of lung adenocarcinoma and adjacent normal tissues. The untransfected A549 cells were divided into blank group, si-NC group （with si-NC transfection） and si-lncRNA group （with silencing of lncRNA TTN-AS1 expression）, with n=5 in each group. The effects of TTN-AS1 silencing on the viability and invasion of A549 cells were detected by CCK8 and Transwell methods. The targeting regulatory effects of TTN-AS1 on miR-519d-3p and miR-519d-3p on MMP2 were determined by dual-luciferase reporter assay, RNA immunoprecipitation test, RT-qPCR and Western blot. RESULTS The expression level of TTN-AS1 in 32 cases of lung adenocarcinoma tissues is notably higher than that in the adjacent normal tissues （P<0.05）. Silencing of TTN-AS1 in A549 cells significantly suppressed the cell viability and invasion. TTN-AS1 negatively regulated the expression of miR-519d-3p via sponging and absorbing miR-519d-3p. MMP2 is the target gene of miR-519d-3p and can be negatively regulated by miR-519d-3p. Overexpression of MMP2 partially reversed the inhibitory effect of TTN-AS1 silencing and miR-519d-3p overexpression on the invasion of A549 cells. CONCLUSION The lncRNA TTN-AS1 is overexpressed in lung adenocarcinoma tissues, and it regulates lung adenocarcinoma A549 cell viability and invasion via miR-519d-3p/MMP2 pathway.     

Assessment of risks to habitat connectivity through the stepping-stone theory: A case study from Shenzhen,China

With the acceleration of urbanization, construction land is gradually expanding, leading to a loss of ecological land. This is increasing the risk of connectivity loss between important natural habitats. In this study, potential ecological corridors and stepping stones under ecological security patterns with different connectivity levels were identified according to the important habitats and resistance surfaces. We analyzed the development potential, vulnerability, and connectivity loss of a fragmented landscape using a support vector machine (SVM), a circuit theory model, and the “exposure-vulnerability-potential loss” framework. Four types of stepping stones that require protection were defined according to the risk structure and level. The results showed that there are 40 important habitats and 50 potential ecological corridors in the ecological security pattern. The SVM results indicated that the possibility of patch development is most sensitive to protection status (−0.923), followed by the slope (−0.770), and distance from the city (−0.210) and roads (−0.147). Additionally, the impact of railways (0.056) and night-time light (0.092) was limited. Elevation (0.267) had a positive effect on development. With an increased security level, the proportion of middle- and high-risk stepping stones gradually decreased, which means that the risk of connectivity loss under urban development can be reduced by creating a security pattern with a higher level and including more stepping-stone patches. Connectivity loss risk management can provide a reference for ecological control line revision and land use planning.     

Where to construct new urban green spaces to be at the recommended distance from users and to complement existing ones? A study in five cities of northern Portugal

The proximity to urban green spaces (UGS) is important to facilitate their use by citizens, mainly by the most vulnerable groups in society who are usually unable to walk long distances. Furthermore, there are several ecosystem services from which the population can only benefit if UGS are close. In addition to being important to identify areas without a UGS nearby, it is also important to indicate possible locations for a new UGS, thus avoiding wasting resources by overlapping coverage areas or leaving gaps in coverage. It is also important to understand among all possible locations where the construction of a UGS is most needed. The present study aims (i) to identify possible locations for future UGS and order them by construction priority, (ii) to develop and test a methodology for decision-makers and urban planners and (iii) to contribute to increasing and developing UGS in cities. Considering residential areas, the results reveal a deficiency in UGS coverage in all the cities studied, as only 43.86–57.09% of residential areas have a UGS within 300 m. Construction of proposed UGS will lead to a 12.30–26.15% increase in coverage. The proposed methodology needs further improvement, but it can be a valuable tool for urban planners and decision-makers, and can encourage the construction of more UGS.     

Effects of marine fungal metabolites 1386A from the South China Sea on proliferation,apoptosis and mitochondrial membrane potential in gastric cancer cell line MCG-803

AIM: To investigate the effects of marine fungal metabolites 1386A from the South China Sea(1386A) on gastric cancer cell line MCG-803.METHODS: The effects of 1386A on cell viability and cell growth were detected by MTT method and colony-forming assay, respectively. The changes of cell cycle, cell apoptosis and mitochondrial membrane potential were determined by flow cytometry.RESULTS: The IC₅₀ of cell viability on MCG-803 cells after treated with 1386A for 24 h, 48 h and 72 h was 29.70 μmol/L, 14.07 μmol/L or 13.47 μmol/L, respectively. The IC₅₀ of the cell growth on MCG-803 cells after treated with 1386A for 48 h was 3.27 μmol/L. After treated with 1386A for 48 h, the MCG-803 cells in S stage was increased from 25.6% to 56.8%. After treated with 1386A for 24 h, the early apoptosis of MCG-803 cells was increased from 3.34% to 8.39%, and the mitochondrial membrane potential of MCG-803 cells was decreased by 56.26%.CONCLUSION: 1386A has an inhibitory effect on the cell growth in MCG-803 cells through the mitochondrial pathway.