Rho/SRF Inhibitor Modulates Mitochondrial Functions.

CCG-1423 is a Rho A pathway inhibitor that has been reported to inhibit Rho/SRF-mediated transcriptional regulation. Serum response factor and its cofactors, which include ternary complex factors and myocardin-related transcription factors, regulate various cellular functions. In this study, we observed that CCG-1423 modulates the mitochondrial functions. The effect of this small molecule drug was determined by measuring mitochondrial function using an XFe96 Analyzer and an Oxygraph 2k (O2k) high-resolution respirometer. CCG-1423 treatment significantly reduced oxidative phosphorylation in a dose-dependent manner. However, CCG-1423 increased the glycolytic rate. We also observed that histone 4 at lysine-16 underwent hyperacetylation with the treatment of this drug. Immunolabeling with F-actin and MitoTracker revealed the alteration in the actin cytoskeleton and mitochondria. Taken together, our findings highlight a critical role of CCG-1423 in inhibiting the transcription of SRF/p49 and PGC-1α, ß, resulting in the downregulation of mitochondrial genes, leading to the repression of mitochondrial oxidative phosphorylation and overall ATP reduction. This study provides a better understanding of the effects of CCG-1423 on mitochondria, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives.

Polyphenol effects on CuO-nanoparticle-mediated DNA damage, reactive oxygen species generation, and fibroblast cell death.

The ability of ten polyphenolic antioxidants to prevent CuO nanoparticle (NPCuO) and H2O2-mediated DNA damage and cytotoxicity was investigated. Five of the polyphenols (MEPCA, PREGA, MEGA, ECG, and EGCG) prevent NPCuO/H2O2-mediated DNA damage (IC50 values of 7.5-800 µM), three have no effect (PCA, VA, and EC), and two (GA and EGC) result in increased DNA damage. Most polyphenols had similar antioxidant/prooxidant activity in the presence of NPCuO or free copper ions. Electron paramagnetic resonance (EPR) spectroscopy of reactive oxygen species (ROS) generated by NPCuO/H2O2 in the presence of representative polyphenols correlate with results of DNA damage studies: in the presence of NPCuO/H2O2, MEPCA prevents ROS formation, VA has no effect on ROS levels, and EGC increases ROS levels. EPR results with CuO nanoparticles washed to remove dissolved copper in solution (wCuO) in the presence of H2O2/ascorbate suggest that MEPCA prevents ROS formation on the nanoparticle surface in addition to preventing ROS formation from dissolved copper. In mouse fibroblast (L929) cells, combining NPCuO with H2O2 results in significantly greater cytotoxicity than observed for either component alone. After 3 h incubation with MEPCA or MEGA, the viability loss in L929 cells induced by NPCuO/H2O2 challenge was significantly rescued at physiologically relevant polyphenol levels (1 µM). These studies show that polyphenols can protect DNA and inhibit cytotoxicity generated by NPCuO under oxidative stress conditions.

Alternative Splicing Increases Sirtuin Gene Family Diversity and Modulates Their Subcellular Localization and Function.

Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. There are seven sirtuin genes in humans, each consists of multiple exons that are likely to undergo alternative splicing. Our aim was to characterize the effect of alternative splicing on the sirtuin genes. Here, we report the identification of 23 human sirtuin isoforms, most of which were not previously reported. Five of the sirtuin genes had more than one isoform, whereas sirtuin-6 had nine isoforms. Exon skipping was the main event. Most of the sirtuin isoforms were deficient in parts of the protein domains, including the catalytic domain, the N- or C-terminus, nuclear localization signal or mitochondrial targeting signal. The domain loss caused potential structural changes. Three SIRT1 isoforms had a differential effect on the mitochondrial oxygen consumption rate. Age-related changes in the expression of SIRT1 isoforms were observed in the human heart in fetus, adults, and very old individuals. We also identified 15 sirtuin isoforms in mice. Our data indicate that alternative splicing increases sirtuin gene diversity and may modulate subcellular localization and function, thereby adding complexity to the gene regulation of mitochondrial respiration, metabolism, and cardiac function during maturation and aging.

Impressively Printing Patterns of Gold and Silver Nanoparticles.

The optical and chemical properties of gold and silver nanoparticles make them useful for many applications, including surface enhanced spectroscopy-based biosensors, photostable colorants, enhanced photovoltaics, and nanoscale optical elements. We report a simple technique to generate patterns of gold and silver nanoparticles with controlled shape and shape-dependent optical properties using metal stamps to impress them onto a glass substrate or flexible polymers. The pressure flattens the nanoparticles, converting initially spherical nanoparticles into discs with reduced height and increased diameter. This deformation causes their localized surface plasmon resonance wavelength to red-shift. Nanoparticles were characterized by electron microscopy, atomic force microscopy, and dark field optical scattering spectroscopy. The deformed nanoparticle patterns had a lateral resolution limited by the nanoparticle diameter (single particles are partly flattened only where they contact the stamp). The method also (i) transfers the stamp's topography, with smooth stamps generating flattened nanoparticles with uniform height, and small changes in stamp height are evident in the nanoparticle height and scattering wavelength, and (ii) allows facile removal of undeformed nanoparticles using scotch tape, and patterns of deformed nanoparticles can be transferred to a thin polymer-film. The patterning process is simple and inexpensive. It can be performed by hand for demonstrations or artistic applications, with controlled force for plasmonics research, and potentially automated on reel-to-reel presses for large scale production.

Reactive oxygen species generation by copper(II) oxide nanoparticles determined by DNA damage assays and EPR spectroscopy.

Copper(II) oxide nanoparticles (NPCuO) have many industrial applications, but are highly cytotoxic because they generate reactive oxygen species (ROS). It is unknown whether the damaging ROS are generated primarily from copper leached from the nanoparticles, or whether the nanoparticle surface plays a significant role. To address this question, we separated nanoparticles from the supernatant containing dissolved copper, and measured their ability to damage plasmid DNA with addition of hydrogen peroxide, ascorbate, or both. While DNA damage from the supernatant (measured using an electrophoresis assay) can be explained solely by dissolved copper ions, damage by the nanoparticles in the presence of ascorbate is an order of magnitude higher than can be explained by dissolved copper and must, therefore, depend primarily upon the nanoparticle surface. DNA damage is time-dependent, with shorter incubation times resulting in higher EC50 values. Hydroxyl radical (•OH) is the main ROS generated by NPCuO/hydrogen peroxide as determined by EPR measurements; NPCuO/hydrogen peroxide/ascorbate conditions generate ascorbyl, hydroxyl, and superoxide radicals. Thus, NPCuO generate ROS through several mechanisms, likely including Fenton-like and Haber-Weiss reactions from the surface or dissolved copper ions. The same radical species were observed when NPCuO suspensions were replaced with the supernatant containing leached copper, washed NPCuO, or dissolved copper solutions. Overall, NPCuO generate significantly more ROS and DNA damage in the presence of ascorbate than can be explained simply from dissolved copper, and the NPCuO surface must play a large role.

Tuning Localized Surface Plasmon Resonance Wavelengths of Silver Nanoparticles by Mechanical Deformation.

We describe a simple technique to alter the shape of silver nanoparticles (AgNPs) by rolling a glass tube over them to mechanically compress them. The resulting shape change in turn induces a red-shift in the localized surface plasmon resonance (LSPR) scattering spectrum and exposes new surface area. The flattened particles were characterized by optical and electron microscopy, single nanoparticle scattering spectroscopy, and surface enhanced Raman spectroscopy (SERS). AFM and SEM images show that the AgNPs deform into discs; increasing the applied load from 0 to 100 N increases the AgNP diameter and decreases the height. This deformation caused a dramatic red shift in the nanoparticle scattering spectrum and also generated new surface area to which thiolated molecules could attach as evident from SERS measurements. The simple technique employed here requires no lithographic templates and has potential for rapid, reproducible, inexpensive and scalable tuning of nanoparticle shape, surface area, and resonance while preserving particle volume.

Removal of molecular adsorbates on gold nanoparticles using sodium borohydride in water.

The mechanism of sodium borohydride removal of organothiols from gold nanoparticles (AuNPs) was studied using an experimental investigation and computational modeling. Organothiols and other AuNP surface adsorbates such as thiophene, adenine, rhodamine, small anions (Br(-) and I(-)), and a polymer (PVP, poly(N-vinylpyrrolidone)) can all be rapidly and completely removed from the AuNP surfaces. A computational study showed that hydride derived from sodium borohydride has a higher binding affinity to AuNPs than organothiols. Thus, it can displace organothiols and all the other adsorbates tested from AuNPs. Sodium borohydride may be used as a hazard-free, general-purpose detergent that should find utility in a variety of AuNP applications including catalysis, biosensing, surface enhanced Raman spectroscopy, and AuNP recycle and reuse.

Inner filter effect on surface enhanced Raman spectroscopic measurement.

Presented herein is a combined experimental and computational study of the gold nanoparticle (AuNP) inner filter effect on surface enhanced Raman spectroscopic (SERS) measurements. Using a bianalyte strategy in which dithiopurine (DTP) and ethanol were employed as the model analytes, we demonstrated that AuNPs enhance DTP's Raman signal but attenuate ethanol's Raman intensity. Combined time-resolved UV-vis and Raman measurements showed that AuNP aggregation has significant and an exactly opposite impact on the AuNP inner filter effect and SERS enhancement. This research provides critical new insights regarding SERS signal variation and offers a simple methodology for reliable determination of the SERS enhancement factors.