Localisation Super-resolution Imaging Using Germanium Quantum Dots.

PhDNovel fluorescent quantum dots of small size, tunable light emission wavelength and high compatibility with biological systems are of great significance to light microscopy super-resolution imaging. In this thesis, colloidal germanium quantum dots of 3.8 nm size have been investigated as a novel fluorescent probe for cell imaging. Two single molecule localisation super-resolution methods were explored: one utilised QDs blinking statistics and the other one was based on intrinsic QDs size dispersion. We found that the blinking super-resolution strategy which combined the usage of blinking QDs and spinning disk confocal imaging has led to less than seven minutes collection time for 2000 image frames. High precision temporal separation of single molecules has been achieved on Ge QDs and CdSe QDs labelled fixed Hela cell. The spectroscopic super-resolution strategy that combined the usage of size dependent light emission QDs and spectra imaging, resulted in a 1.6 seconds data acquisition time. Spectroscopic separation and high precision single molecule localisation has been demonstrated using Ge QDs and CdSe QDs labelled fixed Hela cell samples. We compared various localisation algorithms when applied to the two superresolution methods we studied. We found that they did not work well with our data. Consequently, we developed two MATLAB-based localisation algorithms. The first algorithm used the independent component analysis (ICA) model to analyse the blinking stochastic imaging data, whilst the other used the Gaussian mixed model (GMM) to analyse the spectroscopic separation imaging data. 6 Contents We also conducted comparative toxicity tests of these novel Ge QDs with a typical of-the-shelf system. The cell toxicity of Ge QDs was found to be less than that of CdSe/ZnS QDs. For instance, 25 nM Ge QDs in 1 mL Hela cell solution did not cause observable cells apoptosis in 24 hours. It caused 15% cells apoptosis after 3 days, rather than 35% for CdSe QDs at the same concentration. In addition, long term live cell imaging with QDs revealed that Ge QDs had not significantly changed cellular morphology within a 90 hour period.China Scholarship Council and Queen Mary, University of Londo

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins α5β1 and vβ3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins α5β1 and αvβ3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both α5β1 and αvβ3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was α5β1 and αvβ3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), α5β1, and αvβ3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress

Overexpressing the Sedum alfredii Cu/Zn Superoxide Dismutase Increased Resistance to Oxidative Stress in Transgenic Arabidopsis

Superoxide dismutase (SOD) is a very important reactive oxygen species (ROS)-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD), from Sedum alfredii, a cadmium (Cd)/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT) plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2) and superoxide radicals (O2•-). The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress

HOXC6 promotes migration, invasion and proliferation of esophageal squamous cell carcinoma cells via modulating expression of genes involved in malignant phenotypes

Background HOXC6 is a member of the HOX gene family. The elevated expression of this gene occurs in prostate and breast cancers. However, the role of HOXC6 in esophageal squamous cell carcinoma (ESCC) remains largely uninvestigated. Methods The expression of HOXC6 was examined by immunohistochemistry, quantitative real-time PCR and immunoblotting assays. The lentivirus-mediated expression of HOXC6 was verified at mRNA and protein levels. Wound healing and Matrigel assays were performed to assess the effect of HOXC6 on the migration and invasion of cancer cells. The growth curving, CCK8, and colony formation assays were utilized to access the proliferation capacities. RNA-seq was performed to evaluate the downstream targets of HOXC6. Bioinformatic tool was used to analyze the gene expression. Results HOXC6 was highly expressed in ESCC tissues. HOXC6 overexpression promoted the migration, invasion, and proliferation of both Eca109 and TE10 cells. There were 2,155 up-regulated and 759 down-regulated genes in Eca109-HOXC6 cells and 95 up-regulated and 47 down-regulated genes in TE10-HOXC6 cells compared with the results of control. Interestingly, there were only 20 common genes, including 17 up-regulated and three down-regulated genes with similar changes upon HOXC6 transfection in both cell lines. HOXC6 activated several crucial genes implicated in the malignant phenotype of cancer cells. Discussion HOXC6 is highly expressed in ESCC and promotes malignant phenotype of ESCC cells. HOXC6 can be used as a new therapeutic target of ESCC

Spectroscopic super-resolution fluorescence cell imaging using ultra-small Ge quantum dots

QMUL/CSC scholarship (2011611045); BBSRC grant (BB/J001473/1)

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging

Phenotypic and Comparative Transcriptome Analysis of Different Ploidy Plants in Dendrocalamus latiflorus Munro

Elucidating the differences in gene expression profiles of plants with different ploidy levels and how they affect phenotypic traits is vital to allow genetic improvement of plants such as Ma bamboo (Dendrocalamus latiflorus Munro). We previously obtained triploid (2n = 3X = 36), hexaploid (2n = 6X = 72), and dodecaploid (2n = 12X = 144) Ma bamboo plants from embryogenic callus by anther culturing. Phenotypic differences between these plants appeared to be correlated with differences in ploidy. Here, we performed transcriptome profiling and sequencing of anther-regenerated plants and F1 seedlings of different ploidy levels using RNA-Seq technology. Pair-wise comparisons of the four resulting libraries revealed 8,396 differentially expressed genes. These differentially expressed genes were annotated, functionally classified, and partially validated. We found that the chromosome doubling led to substantially up- or down-regulation of genes that were involved in cell growth and differentiation; the polyploidy levels altered the anatomical, physiological and growth characteristics, such as leaf thickness, fusoid cell and stomatal size, shoot number, photosynthesis and respiration rate and so on. Additionally, two candidate genes, EXPB3 and TCP with potenitial regulatory roles in cell division and differentiation, were identified through gene coexpresseion network analysis. These results highlight the significance of potential applications of polyploidy, and provide valuable information for the genetic breeding of bamboo species

Angular Trajectory Design for MR-SPS Using Bezier Shaping Approach

Solar power satellite (SPS) is a kind of large-scale on-orbit servicing spacecraft collecting solar energy in space and transmitting energy to the earth. The solar arrays of the SPS must point to the sun to collect enough solar energy and the antenna must point to the rectenna on the ground to transmit energy. But due to the limitation of the control effort, accurate solar and earth orientation may not be achieved. This paper focuses on the MR-SPS, and establishes the attitude kinematics and dynamics model of a MW-level MR-SPS. Angular trajectory based on Bezier shaping approach is generated at different time of a year to satisfy the control constraints. The simulation results demonstrate the effectiveness of the proposed method. Even if the control torque is limited to a small amount, the optimal angular trajectory can still ensure high average energy receiving efficiency

Viral infection and immunity view of SARS-CoV-2 using RBD-assembled DNA Soccer-ball Framework

Elevated understanding of the viral infection process contributes to development of neutralizing agents and vaccine to combat infectious diseases. Although crystal structure of single SARS-CoV-2 spike/RBD and host receptor ACE2 is known, the viral attachment and immune response initiated by numbers and distribution patterns of natural spikes on SARS-CoV-2 are still obscure. Leveraging a ~74 nm DNA soccer-ball framework (DSF), we developed an aptamer-guided SARS-CoV-2 RBD precisely assembly strategy, thereby exploring the viral infection and immune response in specific numbers and distributions of RBDs. Thirty evenly distributed RBDs on DSF could achieve sufficient binding affinity against host cell (Kd of 122.2 pM), whereas 60 evenly distributed RBDs on DSF could bind to host cell rapidly (Ka of 0.845 min-1). While RBDs in centralized manner compared to evenly distribution facilitated higher and faster binding to host. Moreover, evenly distributed 20 RBDs on DSF achieved up to 88% immunity elicitation of macrophage cells. Overall, this strategy provides a prospective direction for the assembly of virus-like particles based on DNA origami, thereby facilitating understanding of viral infection and efficient vaccine design

A Multi-Strategy Improved Arithmetic Optimization Algorithm

To improve the performance of the arithmetic optimization algorithm (AOA) and solve problems in the AOA, a novel improved AOA using a multi-strategy approach is proposed. Firstly, circle chaotic mapping is used to increase the diversity of the population. Secondly, a math optimizer accelerated (MOA) function optimized by means of a composite cycloid is proposed to improve the convergence speed of the algorithm. Meanwhile, the symmetry of the composite cycloid is used to balance the global search ability in the early and late iterations. Thirdly, an optimal mutation strategy combining the sparrow elite mutation approach and Cauchy disturbances is used to increase the ability of individuals to jump out of the local optimal. The Rastrigin function is selected as the reference test function to analyze the effectiveness of the improved strategy. Twenty benchmark test functions, algorithm time complexity, the Wilcoxon rank-sum test, and the CEC2019 test set are selected to test the overall performance of the improved algorithm, and the results are then compared with those of other algorithms. The test results show that the improved algorithm has obvious advantages in terms of both its global search ability and convergence speed. Finally, the improved algorithm is applied to an engineering example to further verify its practicability