In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich
strand of the cis-regulatory element in the BCL2 promoter element
is highly dynamic in nature and can form either an i-motif or a flexible
hairpin. Under physiological conditions these two secondary DNA structures
are found in an equilibrium mixture, which can be shifted by the addition
of small molecules that trap out either the i-motif (IMC-48) or the
flexible hairpin (IMC-76). In cellular experiments we demonstrate
that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects
are antagonistic. In this contribution we have identified a transcriptional
factor that recognizes and binds to the BCL2 i-motif
to activate transcription. The molecular basis for the recognition
of the i-motif by hnRNP LL is determined, and we demonstrate that
the protein unfolds the i-motif structure to form a stable single-stranded
complex. In subsequent experiments we show that IMC-48 and IMC-76
have opposite, antagonistic effects on the formation of the hnRNP
LL–i-motif complex as well as on the transcription factor occupancy
at the BCL2 promoter. For the first time we propose
that the i-motif acts as a molecular switch that controls gene expression
and that small molecules that target the dynamic equilibrium of the
i-motif and the flexible hairpin can differentially modulate gene
expression.
It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.
Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (K(d)(app) = 8.5 nM) anti-human E-selectin (CD62E) F(ab')(2) fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol-disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab')(2) fragments. The purified CLIO-F(ab')(2) conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1 beta expressed E-selectin and showed a 100-200 times higher binding of CLIO particles (83-104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30-40 ms in cell precipitates. Cells did not bind/internalize CLIO-F(ab')(2) conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1 beta. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1 beta and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.
Two-dimensional (2D) semiconductors hold promises for electronic and optoelectronic applications due to their outstanding electrical and optical properties. Despite a short research history, a wide range of ‘proof-of-concept’ devices based on 2D materials have been demonstrated, highlighting their impact in advanced technology. Here we review the unique properties 2D semiconducting materials and their applications in terms of electronic and optoelectronic devices. We summarize all the engineering issues in 2D devices, including material quality, dielectric, and contacts. We also discuss recent advances of 2D semiconductor devices in electronic and optoelectronic applications. This review would help to understand superior performance and multifunctions of 2D semiconductor devices and guide us toward new device applications of 2D semiconductors.
OBJECTIVEThyroid hormone accelerates energy expenditure; thus, hypothyroidism is intuitively associated with obesity. However, studies failed to establish such a connection. In brown adipose tissue (BAT), thyroid hormone activation via type 2 deiodinase (D2) is necessary for adaptive thermogenesis, such that mice lacking D2 (D2KO) exhibit an impaired thermogenic response to cold. Here we investigate whether the impaired thermogenesis of D2KO mice increases their susceptibility to obesity when placed on a high-fat diet.RESEARCH DESIGN AND METHODSTo test this, D2KO mice were admitted to a comprehensive monitoring system acclimatized to room temperature (22°C) or thermoneutrality (30°C) and kept either on chow or high-fat diet for 60 days.RESULTSAt 22°C, D2KO mice preferentially oxidize fat, have a similar sensitivity to diet-induced obesity, and are supertolerant to glucose. However, when thermal stress is eliminated at thermoneutrality (30°C), an opposite phenotype is encountered, one that includes obesity, glucose intolerance, and exacerbated hepatic steatosis. We suggest that a compensatory increase in BAT sympathetic activation of the D2KO mice masks metabolic repercussions that they would otherwise exhibit.CONCLUSIONSThus, upon minimization of thermal stress, high-fat feeding reveals the defective capacity of D2KO mice for diet-induced thermogenesis, provoking a paradigm shift in the understanding of the role of the thyroid hormone in metabolism.
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