Modification and Characterization of Fluorescent Conjugated Polymer Nanoparticles for Single Molecule Detection

Single molecule tracking using fluorescent dye or nanoparticle labels has emerged as a useful technique for probing biomolecular processes. Considerable interest arises in the development of nanoparticle labels with brighter fluorescence in order to improve the spatial and temporal resolution of single molecule detection and to facilitate the application of single molecule detection methods to a wider range of intracellular processes. The McNeill laboratory recently reported that conjugated polymer nanoparticles exhibit fluorescence cross-sections roughly 10-100 times higher than other luminescent nanoparticles of similar size, excellent photostability (2.2×108 photons emitted per nanoparticle prior to photobleaching), and saturated emission rates roughly 100 times higher than that of the molecular dyes and more than 1000 times higher than that of colloidal semiconductor quantum dots. One purpose of this graduate research is the development of highly fluorescent, bioconjugated nanoparticle labels based on conjugated polymers for demanding fluorescence applications such as single molecule tracking in live cells. Three surface modification methods (conjugated polymer nanoparticles encapsulated with lipid silica agents, conjugated polymer nanoparticles encapsulated with tetraethyl orthosilicate(TEOS) and hybrid nanoparticles with thiol pendant groups by the Stöber Method (3-mercaptopropyl trimethoxysilane (MPS))) have been developed to protect the conjugated polymer, passivate the nanoparticle surface, and provide a chemical handle for bioconjugation such as nanoparticle encapsulation with alkoxysilanes and Stöber method. After encapsulation, the fluorescence quantum yield of silica-encapsulated nanoparticles is improved by 20% as compared to bare conjugated polymer nanoparticles, while the photostability is improved by a factor of 2, indicating that some protection of the polymer is provided by the encapsulating layer. Another purpose of my research is the manipulation of the photophysics and photochemistry of conjugated polymer nanoparticles based on developing a more complete understanding of the various processes that control or limit nanoparticle brightness and photostability. The results indicate that a combination of photophysical processes including electron transfer to molecular oxygen, energy transfer, and exciton diffusion result in saturation and photobleaching phenomena that currently limit brightness. This study provides the potential methods and strategies aimed at manipulating such processes or limiting their effect on fluorescence brightness. Finally, efficient intra-particle energy transfer has been demonstrated in dye-doped CP nanoparticles, which provides a new strategy for improving nanoparticle fluorescence brightness and photostability, obtaining nanoparticles with red emission to avoid autofluorescence in mammalian tissue, and for designing novel sensitive biosensors based on energy transfer to sensor dyes

Large-scale Molecular Dynamics Simulation with Forward Flux Sampling on Hadoop

Simulating rare events is extremely difficulty and requires massive computational resources and complex data processing workflow, which is determined by the nature of stochastic systems. To help computational scientists discover hard scientific problems in this area, we built a large-scale molecular dynamics simulation framework integrated with forward flux sampling (FFS) technique on Hadoop ecosystem. In this project, we port the customized FFS workflow to underlying MapReduce-based computing pipeline by using dataflow-driven design pattern and Gromacs application. The early works show that our framework is able to provide a scalable, fault-tolerance and efficient rare events simulation environment over varieties of computing infrastructures, while preserving the flexibility of the original scientific application

Enhancing Human Spermine Synthase Activity by Engineered Mutations

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing

MAPK1 promotes the metastasis and invasion of gastric cancer as a bidirectional transcription factor

Background: The Mitogen-activated protein kinase 1 (MAPK1) has both independent functions of phosphorylating histones as a kinase and directly binding the promoter regions of genes to regulate gene expression as a transcription factor. Previous studies have identified elevated expression of MAPK1 in human gastric cancer, which is associated with its role as a kinase, facilitating the migration and invasion of gastric cancer cells. However, how MAPK1 binds to its target genes as a transcription factor and whether it modulates related gene expressions in gastric cancer remains unclear. Results: Here, we integrated biochemical assays (protein interactions and chromatin immunoprecipitation (ChIP)), cellular analysis assays (cell proliferation and migration), RNA sequencing, ChIP sequencing, and clinical analysis to investigate the potential genomic recognition patterns of MAPK1 in a human gastric adenocarcinoma cell-line (AGS) and to uncover its regulatory effect on gastric cancer progression. We confirmed that MAPK1 promotes AGS cells invasion and migration by regulating the target genes in different directions, up-regulating seven target genes (KRT13, KRT6A, KRT81, MYH15, STARD4, SYTL4, and TMEM267) and down-regulating one gene (FGG). Among them, five genes (FGG, MYH15, STARD4, SYTL4, and TMEM267) were first associated with cancer procession, while the other three (KRT81, KRT6A, and KRT13) have previously been confirmed to be related to cancer metastasis and migration. Conclusion: Our data showed that MAPK1 can bind to the promoter regions of these target genes to control their transcription as a bidirectional transcription factor, promoting AGS cell motility and invasion. Our research has expanded the understanding of the regulatory roles of MAPK1, enriched our knowledge of transcription factors, and provided novel candidates for cancer therapeutics

An Infrastructure to Support Data Integration and Curation for Higher Educational Research

The recent challenges for higher education call for research that can offer a comprehensive understanding about the performance and efficiency of higher education institutions in their three primary missions: research, education, and service. In other for this to happen, it is necessary for researchers to have access to a multitude of data sources.However, due to the nature of their academic training, many higher education practitioners do not have access to expertise in working with different data sources. In this work, we describe a design and implementation for an infrastructure that will bring together the tools and the data to provide access to researchers in the field of higher education institutional research. The infrastructure will include integration and curation for data from different sources, embedded statistical environment, high performance computational back-end, and extensibility for future Big Data and unstructured data

A New Digital Image Hiding Algorithm Based on Wavelet Packet Transform and Singular Value Decomposition

The paper presents a new digital image hiding algorithm based on wavelet packets transform and singular value decomposition. The low-frequency sub-band of wavelet packets transform has strong anti-jamming capacity and the singular value has very strong stability. The presented algorithm implements bit plane decomposition on the secret image and wavelet packet decomposition on the carrier image. Then, it hides the bit planes with important information into the singular value matrix of the low frequency coefficient matrix, and also hides the bit planes with secondary information into the remainder sub-band matrix with higher entropy energy. The hiding location is adaptively determined by the carrier image. The experimental results indicate that, the proposed image hiding algorithm has strong robustness and anti-attack, and it also has good invisibility and big capability

Craniofacial Reconstruction via Face Elevation Map Estimation Based on the Deep Convolution Neutral Network

In this study, to achieve the possibility of predicting face by skull automatically, we propose a craniofacial reconstruction method based on the end-to-end deep convolutional neural network. Three-dimensional volume data are obtained from 1447 head CT scans of Chinese people of different ages. The facial and skull surface data are projected onto two-dimensional space to generate a two-dimensional elevation map, and then, use the deep convolution neural network to realize the prediction of skull to face shape in two-dimensional space. The encoder and decoder are composed of first feature extraction through the encoder and then as the input of the decoder to generate the craniofacial restoration image. In order to accurately describe the features of different scales, we adopt an U-shaped codec structure with cross-layer connections. Therefore, the output features are decomposed with the features of the corresponding scales in the encoding stage to achieve the integration of different scales while restoring the feature scales in the compression and decoding stage. Meanwhile, the U-net structures help to avoid the problem of loss of detail features in the downsampling process. We use supervised learning to obtain the prediction model from skull to facial elevation map. Back-projection operation is performed afterwards to generate facial surface data in 3D space. Experiments show that the proposed method in this study can effectively achieve craniofacial reconstruction, and for most part of the face, restoration error is controlled within 2 mm

Efficient biosynthesis of pinosylvin from lignin-derived cinnamic acid by metabolic engineering of Escherichia coli

Abstract Background The conversion of lignin-derived aromatic monomers into valuable chemicals has promising potential to improve the economic competitiveness of biomass biorefineries. Pinosylvin is an attractive pharmaceutical with multiple promising biological activities. Results Herein, Escherichia coli was engineered to convert the lignin-derived standard model monomer cinnamic acid into pinosylvin by introducing two novel enzymes from the wood plant: stilbene synthase from Pinus pinea (PpSTS) and 4-Coumarate-CoA ligase from Populus trichocarpa (Ptr4CL4). The expression of Ptr4CL4 drastically improved the production of pinosylvin (42.5 ± 1.1 mg/L), achieving values 15.7-fold higher than that of Ptr4CL5 (another 4-Coumarate-CoA ligase from Populus trichocarpa) in the absence of cerulenin. By adjusting the expression strategy, the optimized engineered strain produced pinosylvin at 153.7 ± 2.2 mg/L with an extremely high yield of 1.20 ± 0.02 mg/mg cinnamic acid in the presence of cerulenin, which is 83.9% ± 1.17 of the theoretical yield. This is the highest reported pinosylvin yield directly from cinnamic acid to date. Conclusion Our work highlights the feasibility of microbial production of pinosylvin from cinnamic acid and paves the way for converting lignin-related aromatics to valuable chemicals

Efficient Detection Of Viral Transmissions With Next-Generation Sequencing Data

Background: Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Molecular analysis has been frequently used in the study of HCV outbreaks and transmission chains; helping identify a cluster of sequences as linked by transmission if their genetic distances are below a previously defined threshold. However, HCV exists as a population of numerous variants in each infected individual and it has been observed that minority variants in the source are often the ones responsible for transmission, a situation that precludes the use of a single sequence per individual because many such transmissions would be missed. The use of Next-Generation Sequencing immensely increases the sensitivity of transmission detection but brings a considerable computational challenge because all sequences need to be compared among all pairs of samples. Methods: We developed a three-step strategy that filters pairs of samples according to different criteria: (i) a k-mer bloom filter, (ii) a Levenhstein filter and (iii) a filter of identical sequences. We applied these three filters on a set of samples that cover the spectrum of genetic relationships among HCV cases, from being part of the same transmission cluster, to belonging to different subtypes. Results: Our three-step filtering strategy rapidly removes 85.1% of all the pairwise sample comparisons and 91.0% of all pairwise sequence comparisons, accurately establishing which pairs of HCV samples are below the relatedness threshold. Conclusions: We present a fast and efficient three-step filtering strategy that removes most sequence comparisons and accurately establishes transmission links of any threshold-based method. This highly efficient workflow will allow a faster response and molecular detection capacity, improving the rate of detection of viral transmissions with molecular data

The Effect of Cooling Rate on Microstructure and Mechanical Properties of the Zr-4Hf-3Nb (wt%) Alloy

The mechanical properties of Zr-based alloys, such as strength and elongation, are heavily dependent on the cooling rate during heat treatment. Understanding the phase transformation and microstructural evolution in various cooling media can establish the connection between the cooling rate and mechanical properties. The effect of the cooling rate on the phase, microstructure, and tensile properties of Zr-4Hf-3Nb (wt%) alloy is studied in this paper. The results show that the phase composition of the samples transforms from α+β to α+β+ω, and, finally, to α+α’+ω, while the average grain size of α phase decreases from 3.73 μm to 1.96 μm, and the distribution varies from compact to scattering as the cooling rate increases. Hf tends to distribute in β phase, and the slower cooling rate is beneficial to the existence of Hf. The strength and microhardness enhances monotonously, while the elongation ascends first, then decreases as the cooling rate increases. The high strength of water-cooling samples is attributed to the reduction in average grain size and volume fraction of α phase, and the lath α’ martensite and granular ω phase. The fracture pattern of Zr-4Hf-3Nb (wt%) alloy is ductile fracture, and the plasticity gets better with decreasing cooling rate