40 research outputs found
DNA Bending Force Facilitates Z-DNA Formation under Physiological Salt Conditions
Ā© 2022 American Chemical Society.Z-DNA, a noncanonical helical structure of double-stranded DNA (dsDNA), plays pivotal roles in various biological processes, including transcription regulation. Mechanical stresses on dsDNA, such as twisting and stretching, help to form Z-DNA. However, the effect of DNA bending, one of the most common dsDNA deformations, on Z-DNA formation is utterly unknown. Here, we show that DNA bending induces the formation of Z-DNA, that is, more Z-DNA is formed as the bending force becomes stronger. We regulated the bending force on dsDNA by using D-shaped DNA nanostructures. The B-Z transition was observed by single-molecule fluorescence resonance energy transfer. We found that as the bending force became stronger, Z-DNA was formed at lower Mg2+ concentrations. When dsDNA contained cytosine methylations, the B-Z transition occurred at 78 mM Mg2+ (midpoint) in the absence of the bending force. However, the B-Z transition occurred at a 28-fold lower Mg2+ concentration (2.8 mM) in the presence of the bending force. Monte Carlo simulation suggested that the B-Z transition stabilizes the bent form via the formation of the B-Z junction with base extrusion, which effectively releases the bending stress on DNA. Our results clearly show that the bending force facilitates the B-Z transition under physiological salt conditions.N
Determination of the Parameter Sets for the Best Performance of IPS-driven ENLIL Model
Interplanetary scintillation-driven (IPS-driven) ENLIL model was jointly developed by University of California, San Diego
(UCSD) and National Aeronaucics and Space Administration/Goddard Space Flight Center (NASA/GSFC). The model has
been in operation by Korean Space Weather Cetner (KSWC) since 2014. IPS-driven ENLIL model has a variety of ambient
solar wind parameters and the results of the model depend on the combination of these parameters. We have conducted
researches to determine the best combination of parameters to improve the performance of the IPS-driven ENLIL model.
The model results with input of 1,440 combinations of parameters are compared with the Advanced Composition Explorer
(ACE) observation data. In this way, the top 10 parameter sets showing best performance were determined. Finally, the
characteristics of the parameter sets were analyzed and application of the results to IPS-driven ENLIL model was discussed
Cytosine methylation regulates DNA bendability depending on the curvature
Cytosine methylation plays an essential role in many biological processes, such as nucleosome inactivation and regulation of gene expression. The modulation of DNA mechanics may be one of the regulatory mechanisms influenced by cytosine methylation. However, it remains unclear how methylation influences DNA mechanics. Here, we show that methylation has contrasting effects on the bending property of dsDNA depending on DNA curvature. We directly applied bending force on 30 base pairs of dsDNA using a D-shaped DNA nanostructure and measured the degree of bending using single-molecule fluorescence resonance energy transfer without surface immobilization. When dsDNA is weakly bent, methylation increases the stiffness of dsDNA. The stiffness of dsDNA increased by approximately 8% with a single methylation site for 30 bp dsDNA. When dsDNA is highly bent by a strong force, it forms a kink, i.e., a sharp bending of dsDNA. Under strong bending, methylation destabilizes the non-kink form compared with the kink form, which makes dsDNA near the kink region apparently more bendable. However, if the kink region is methylated, the kink form is destabilized, and dsDNA becomes stiffer. As a result, methylation increases the stiffness of weakly bent dsDNA and concurrently can promote kink formation, which may stabilize the nucleosome structure. Our results provide new insight into the effect of methylation, showing that cytosine methylation has opposite effects on DNA mechanics depending on its curvature and methylation location.N
Expression level of human TLR4 rather than sequence is the key determinant of LPS responsiveness
<div><p>To address the role of Toll-like receptor 4 (TLR4) single nucleotide polymorphisms (SNP) in lipopolysaccharide (LPS) recognition, we generated mice that differed only in the sequence of TLR4. We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using <i>in vitro</i> and <i>in vivo</i> assays we found that the expression level rather than the sequence of TLR4 played a larger role in recognition of LPS, especially hypoacylated LPS.</p></div
Reduced responsiveness to hypoacylated LPS by TLR4 SNP primary splenocyte mac/mono populations.
<p>Percent of mac/mono cells that are TNF+ in response to 1000 ng/ml PA LPS, YP LPS or MPL, top row, or percent TNF+ corrected by the percent TNF+ in response to CpG, bottom row. * above genotypes shows results compared to B6 whereas ā is compared to KO. Data for huTLR4<sup>WT</sup> in response to MPL were combined for 1-way ANOVA since the 4-copy line was only tested once and our previous studies did not reveal any differences between the 2- and 4-copy lines.</p
TLR4 expression correlates with copy number.
<p>(A) BMDM from each of the genotypes indicated above plots were stained with anti-huTLR4 clone HTA125 or isotype control. Boxed number in each plot shows ĪMFI of TLR4 (red histogram for huTLR4, blue histogram in B for muTLR4) vs. isotype control (filled grey histogram). (B) B6 mouse BMDM stained with anti-muTLR4. (C) Each symbol represents a separate BMDM preparation. All cells express huMD-2; red squares depict huTLR4<sup>WT</sup>, light blue triangles huTLR4<sup>D299G</sup> (299), and dark blue inverted triangles huTLR4<sup>D299G+T399I</sup> (399). X (KO) does not express TLR4. Open symbols have lower copy numbers than closed symbols. Brackets show significant pair-wise comparisons using 1-way ANOVA followed by Bonferroniās Multiple Comparison test. (D) Relative TLR4 mRNA expression in BMDM from each genotype, including WT B6 BMDM (average of 3 separate BMDM preparations). The same primers were used for human and mouse TLR4, and expression relative to Ć-actin is shown. Plotted are the means +/- SD. 1-way ANOVA results for D are shown to the right. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001, ns = not significant.</p
Cytokine responses in supernatants from BMDM stimulated with indicated LPS.
<p>An equal number (10<sup>6</sup> cells) of BMDM were plated in 96-well plates and stimulated with 1000 ng/ml PA or YP LPS, or 10 ng/ml EC LPS. Cytokines secreted into the supernatants were measured 24-hr later by Luminex. Results from 2 independent BMDM preparations are shown. * above genotypes shows results compared to B6 whereas ā is compared to KO. 1-way ANOVA results for the EC IL-6 responses are shown in the table.</p
Serum cytokine responses to 100 Ī¼g EC LPS 1 + 6 hr post IP injection.
<p>Mice of the indicated genotypes were injected IP with EC LPS and then bled from the retro-orbital sinus 1 hr after injection. At 6 hr, the mice were euthanized for a terminal sample collection. Shown are combined data from 3 experiments, with 1 experiment containing 1 and 6 hr time points whereas the other 2 had either the 1 or 6 hr time point. All genotypes were included in all experiments. Data are plotted on a log scale for easier visualization due to large variation. Line indicates mean for genotype. * above symbols shows results compared to B6 whereas ā is compared to KO.</p
Humanized TLR7/8 Expression Drives Proliferative Multisystemic Histiocytosis in C57BL/6 Mice
<div><p>A humanized <i>TLR7/TLR8</i> transgenic mouse line was engineered for studies using TLR7/8 ligands as vaccine adjuvants. The mice developed a spontaneous immune-mediated phenotype prior to six months of age characterized by runting, lethargy, blepharitis, and corneal ulceration. Histological examination revealed a marked, multisystemic histiocytic infiltrate that effaced normal architecture. The histological changes were distinct from those previously reported in mouse models of systemic lupus erythematosus. When the mice were crossed with <i>MyD88<sup>ā/ā</sup></i> mice, which prevented toll-like receptor signaling, the inflammatory phenotype resolved. Illness may be caused by constitutive activation of human <i>TLR7</i> or <i>TLR8</i> in the bacterial artificial chromosome positive mice as increased TLR7 and TLR8 expression or activation has previously been implicated in autoimmune disease.</p></div
Ninhydrin Loaded Microcapsules for Detection of Natural Free Amino Acid
Natural free amino acids present in plant extracts or tea infusions provide a unique flavor and potential effect on anxiety and blood pressure reduction. Accordingly, quantifying free amino acids in foods has been of interest to food science and analytical research fields. The ninhydrin solution-based assay is a colorimetric method based on the formation and detection of Ruhemannās purple complex. Media-based colorimetric detection requires specialized facilities and personnel; moreover, it can suffer from the interference of the analyte color. In this study, we developed ninhydrin-loaded microcapsules and a simple free amino acids detection procedure, by simply dipping the microcapsules into the analyte solution for 3 min. Among the five tested natural free amino acids, theanine exhibited the highest colorimetric response to microcapsule-based detection, with a limit of detection of 0.826 mM