113 research outputs found
DNA strand exchange and hydrophobic interactions between biomolecules
The role of hydrophobic interactions in DNA strand exchange has been studied using fluorescence-labeled DNA oligomers in a FRET assay. Strand exchange was found to be accelerated in the presence of polyethylene glycol, which provides a crowded and hydrophobic environment possibly mimicking that of the catalytically active recombinase-DNA complexes. Circular dichroism spectroscopy shows that B-DNA conformation is conserved, so the increased rate of exchange is not simply caused by melting of DNA duplexes. A hydrophobic environment increases the base pairing accuracy of DNA strand exchange, which causes mismatched duplexes to quickly be replaced in the presence of matching strands. It is inferred that these effects are caused by a decrease in water activity which weakens the DNA stacking forces, and by favorable hydrophobic interactions between PEG and DNA chains, with the result that DNA breathing and subsequent strand invasion is facilitated.
Linear dichroism and dynamic light scattering were also used to study some other biomolecular systems where hydrophobic interactions are important: lipid membranes, DNA-protein complex, DNA nanoconstructs anchored to membrane surface, and to study fusion of liposomes induced by shearing forces. A DNA hexagon construct was found to adopt different orientations at the membrane surface depending on the number of attached anchors, but the construct itself was inferred to have a metastable shape due to internal flexibility. Finally, an example of assembly of protein subunits to a membrane surface was considered in shape of the ATP synthase system for which we propose that the activation energy of ATP synthesis may be reduced through coupled reactions between three active sites. The results are interesting in more general contexts of methodological improvements for studying biomolecular assembly, including linear dichroism spectroscopy of transmembrane proteins
Mismatch detection in homologous strand exchange amplified by hydrophobic effects
In contrast to DNA replication and transcription where nucleotides are added and matched one by one, homologous recombination by DNA strand exchange tests whole sequences for complementarity, which requires elimination of mismatched yet thermodynamically stable intermediates. To understand the remarkable sequence specificity of homologous recombination, we have studied strand exchange between a 20-mer duplex containing one single mismatch (placed at varied positions) with the matching single strand in presence of poly(ethylene glycol) representing a semi-hydrophobic environment. A FRET-based assay shows that rates and yields of strand exchange from mismatched to matched strands rapidly increase with semi-hydrophobic co-solute concentration, contrasting previously observed general strand exchange accelerating effect of ethyl glycol ethers. We argue that this effect is not caused simply by DNA melting or solvent-induced changes of DNA conformation but is more complex involving several mechanisms. The catalytic effects, we propose, involve strand invasion facilitated by reduced duplex stability due to weakened base stacking (“longitudinal breathing”). Secondly, decreased water activity makes base-pair hydrogen bonds stronger, increasing the relative energy penalty per mismatch. Finally, unstacked mismatched bases (gaps) are stabilized through partly intercalated hydrophobic co-solvent molecules, assisting nucleation of strand invasion at the point of mismatch. We speculate that nature long ago discovered, and now exploits in various enzymes, that sequence recognition power of nucleic acids may be modulated in a hydrophobic environment
Identification of Thioflavin T Binding Modes to DNA: A Structure-Specific Molecular Probe for Lasing Applications
The binding mechanism of thioflavin T (ThT) to DNA was studied using polarized light spectroscopy and fluorescence-based techniques in solutions and in solid films. Linear dichroism measurements showed that ThT binds to DNA duplex by intercalation. Time-resolved fluorescence studies revealed a second binding mode which is the external binding to the DNA phosphate groups. Both binding modes represent the nonspecific type of interactions. The studies were complemented with the analysis of short oligonucleotides having DNA cavities. The results indicate that the interplay between three binding modes-intercalation, external binding, and binding inside DNA cavities-determines the effective fluorescence quantum yield of the dye in the DNA structures. External binding was found to be responsible for fluorescence quenching because of energy transfer between intercalated and externally bound molecules. Finally, amplified spontaneous emission (ASE) was successfully generated in the ThT-stained films and used for detecting different DNA structures. ASE measurements show that ThT-stained DNA structures can be used for designing bioderived microlasers
Bayesian updating of soil-water character curve parameters based on the monitor data of a large-scale landslide model experiment
It is important to determine the soil-water characteristic curve (SWCC) for analyzing landslide seepage under varying hydrodynamic conditions. However, the SWCC exhibits high uncertainty due to the variability inherent in soil. To this end, a Bayesian updating framework based on the experimental data was developed to investigate the uncertainty of the SWCC parameters in this study. The objectives of this research were to quantify the uncertainty embedded within the SWCC and determine the critical factors affecting an unsaturated soil landslide under hydrodynamic conditions. For this purpose, a large-scale landslide experiment was conducted, and the monitored water content data were collected. Steady-state seepage analysis was carried out using the finite element method (FEM) to simulate the slope behavior during water level change. In the proposed framework, the parameters of the SWCC model were treated as random variables and parameter uncertainties were evaluated using the Bayesian approach based on the Markov chain Monte Carlo (MCMC) method. Observed data from large-scale landslide experiments were used to calculate the posterior information of SWCC parameters. Then, 95% confidence intervals for the model parameters of the SWCC were derived. The results show that the Bayesian updating method is feasible for the monitoring of data of large-scale landslide model experiments. The establishment of an artificial neural network (ANN) surrogate model in the Bayesian updating process can greatly improve the efficiency of Bayesian model updating
Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties
Simple sequence repeats (SSR) – also known as microsatellites – have been used extensively in genetic analysis, fine mapping, quantitative trait locus (QTL) mapping, as well as marker-assisted selection (MAS) breeding and other techniques. Despite a plethora of studies reporting that perfect SSRs with stable motifs and flanking sequences are more efficient for genetic research, the lack of a high throughput technology for SSR genotyping has limited their use as genetic targets in many crops. In this study, we developed a technology called Target SSR-seq that combined the multiplexed amplification of perfect SSRs with high throughput sequencing. This method can genotype plenty of SSR loci in hundreds of samples with highly accurate results, due to the substantial coverage afforded by high throughput sequencing. We also detected 844 perfect SSRs based on 182 resequencing datasets in cucumber, of which 91 SSRs were selected for Target SSR-seq. Finally, 122 SSRs, including 31 SSRs for varieties identification, were used to genotype 382 key cucumber varieties readily available in Chinese markets using our Target SSR-seq method. Libraries of PCR products were constructed and then sequenced on the Illumina HiSeq X Ten platform. Bioinformatics analysis revealed that 111 filtered SSRs were accurately genotyped with an average coverage of 1289× at an extremely low cost; furthermore, 398 alleles were observed in 382 cucumber cultivars. Genetic analysis identified four populations: northern China type, southern China type, European type, and Xishuangbanna type. Moreover, we acquired a set of 16 core SSRs for the identification of 382 cucumber varieties, of which 42 were isolated as backbone cucumber varieties. This study demonstrated that Target SSR-seq is a novel and efficient method for genetic research
Lipidomics Reveals Multiple Pathway Effects of a Multi-Components Preparation on Lipid Biochemistry in ApoE*3Leiden.CETP Mice
Background: Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses. Methodology/Principal Findings: ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p <0.001), CETP levels (-31%,
Choose your cell model wisely: The in vitro nanoneurotoxicity of differentially coated iron oxide nanoparticles for neural cell labeling
Currently, there is a large interest in the labeling of neural stem cells (NSCs) with iron oxide nanoparticles (IONPs) to allow MRI-guided detection after transplantation in regenerative medicine. For such biomedical applications, excluding nanotoxicity is key. Nanosafety is primarily evaluated in vitro where an immortalized or cancer cell line of murine origin is often applied, which is not necessarily an ideal cell model. Previous work revealed clear neurotoxic effects of PMA-coated IONPs in distinct cell types that could potentially be applied for nanosafety studies regarding neural cell labeling. Here, we aimed to assess if DMSA-coated IONPs could be regarded as a safer alternative for this purpose and how the cell model impacted our nanosafety optimization study. Hereto, we evaluated cytotoxicity, ROS production, calcium levels, mitochondrial homeostasis and cell morphology in six related neural cell types, namely neural stem cells, an immortalized cell line and a cancer cell line from human and murine origin. The cell lines mostly showed similar responses to both IONPs, which were frequently more pronounced for the PMA-IONPs. Of note, ROS and calcium levels showed opposite trends in the human and murine NSCs, indicating the importance of the species. Indeed, the human cell models were overall more sensitive than their murine counterpart. Despite the clear cell type-specific nanotoxicity profiles, our multiparametric approach revealed that the DMSA-IONPs outperformed the PMA-IONPs in terms of biocompatibility in each cell type. However, major cell type-dependent variations in the observed effects additionally warrant the use of relevant human cell models.status: publishe
DNA strand exchange and hydrophobic interactions between biomolecules
The role of hydrophobic interactions in DNA strand exchange has been studied using fluorescence-labeled DNA oligomers in a FRET assay. Strand exchange was found to be accelerated in the presence of polyethylene glycol, which provides a crowded and hydrophobic environment possibly mimicking that of the catalytically active recombinase-DNA complexes. Circular dichroism spectroscopy shows that B-DNA conformation is conserved, so the increased rate of exchange is not simply caused by melting of DNA duplexes. A hydrophobic environment increases the base pairing accuracy of DNA strand exchange, which causes mismatched duplexes to quickly be replaced in the presence of matching strands. It is inferred that these effects are caused by a decrease in water activity which weakens the DNA stacking forces, and by favorable hydrophobic interactions between PEG and DNA chains, with the result that DNA breathing and subsequent strand invasion is facilitated. Linear dichroism and dynamic light scattering were also used to study some other biomolecular systems where hydrophobic interactions are important: lipid membranes, DNA-protein complex, DNA nanoconstructs anchored to membrane surface, and to study fusion of liposomes induced by shearing forces. A DNA hexagon construct was found to adopt different orientations at the membrane surface depending on the number of attached anchors, but the construct itself was inferred to have a metastable shape due to internal flexibility. Finally, an example of assembly of protein subunits to a membrane surface was considered in shape of the ATP synthase system for which we propose that the activation energy of ATP synthesis may be reduced through coupled reactions between three active sites. The results are interesting in more general contexts of methodological improvements for studying biomolecular assembly, including linear dichroism spectroscopy of transmembrane proteins
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