As the leading nanodevice candidate, single-walled carbon nanotubes (SWNTs) have potential therapeutic applications in gene therapy and novel drug delivery. We found that SWNTs can inhibit DNA duplex association and selectively induce human telomeric i-motif DNA formation by binding to the 5-end major groove under physiological conditions or even at pH 8.0. SWNT binding to telomeric DNA was studied by UV melting, NMR, S1 nuclease cleavage, CD, and competitive FRET methods. These results suggest that SWNTs might have the intriguing potential to modulate human telomeric DNA structures in vivo, like biologically relevant B-A and B-Z DNA transitions, which is of great interest for drug design and cancer therapy. Since the discovery of the DNA i-motif, the formation and biological function of this unique structure have attracted much attention, and significant progress in characterizing the i-motif has been made in recent years (1, 2). The biological importance of the intramolecular i-motif is evidenced by its involvement in human telomeric and centromeric DNA structures and RNA intercalated structures, and by the discovery of several proteins that bind specifically to C-rich telomeric DNA fragments capable of forming i-motifs (2). Along with human telomeric G-quadruplex DNA, the i-motif has been an attractive drug target for cancer chemotherapy and for modulation of gene transcription (2-9). Human telomeres consist of tandem repeats of the double-stranded DNA sequence (5Ј-TTAGGG):(5Ј-CCCTAA) (3-9). The G-rich strand can form a four-stranded G-quadruplex consisting of G-quartets, whereas its complementary C-rich strand may adopt i-motif structures with intercalated C⅐C ϩ base pairs ( Fig. 1). Significantly, G-quadruplex formation was shown to inhibit the activity of telomerase, an enzyme that stabilizes chromosome ends by adding tandem telomeric DNA repeats to the 3Ј single-stranded overhangs (4,5,10). In contrast to the majority of normal human somatic cells, most tumor cells express telomerase, and telomerase expression is essential for their indefinite proliferative capacities. Therefore, the therapeutic targeting of telomerase activity has been considered a promising approach to cancer therapy (4, 5, 10). Several small molecules have been shown to efficiently inhibit telomerase activity through the stabilization of G-quadruplex DNA (4, 5). Only two molecules that can stabilize both G-quadruplex and i-motif DNA have been identified (11,12). To our knowledge, a ligand that can selectively stabilize i-motif DNA but not G-quadruplex DNA has not been reported.As the leading nanodevice candidate, single-walled carbon nanotubes (SWNTs) have potential applications ranging from gene therapy to novel drug delivery to membrane separation (13-17). B-Z DNA transition has been achieved on the surfaces of SWNTs (18). Recently we reported that SWNTs bind to the DNA major groove and can induce a sequence-dependent B-A DNA transition (19). In the present study we show that SWNTs can selectively stabilize human telomeric i-motif DN...

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nanodevice applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40°C by SWNTs but no change for AT-DNA. SWNTs can induce B–A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B–A transition in solution and they bind to the DNA major groove with GC preference.

The increasing worldwide demand for carbon nanotubes (CNTs) and increasing concern regarding how to safely develop and use CNTs are requiring a low-cost, simple, and highly sensitive CNT detection assay for toxicological evaluation and environmental monitoring. However, this goal is still far from being achieved. All the current CNT detection techniques are not applicable for automation and field analysis because they are dependent on highly expensive special instruments and complicated sample preparation. On the basis of the capability of single-walled carbon nanotubes (SWNTs) to specifically induce human telomeric i-motif formation, we design an electrochemical DNA (E-DNA) sensor that can distinguish single- and multiwalled carbon nanotubes both in buffer and in cell extracts. The E-DNA sensor can selectively detect SWNTs with a direct detection limit of 0.2 ppm and has been demonstrated in cancer cell extracts. To the best of our knowledge, this is the first demonstration of a biosensing technique that can distinguish different types of nanotubes. Our work will provide new insights into how to design a biosensor for detection of carbon nanotubes.

Summary Background The PINK1-Parkin pathway is known to play important roles in regulating mitochondria dynamics, motility, and quality control. Activation of this pathway can be triggered by a variety of cellular stressor signals that cause mitochondrial damage. How this pathway senses different levels of mitochondrial damage and mediates cell fate decisions accordingly is incompletely understood. Results Here, we present evidence that PINK1-Parkin has both cytoprotective and pro-apoptotic functions. PINK1-Parkin operates as a molecular switch to dictate cell fate decisions in response to different cellular stressors. Cells exposed to severe and irreparable mitochondrial damage agents such as valinomycin can undergo PINK1-Parkin-dependent apoptosis. The proapoptotic response elicited by valinomycin is associated with the degradation of Mcl-1. PINK1 directly phosphorylates Parkin at Ser65 of its Ubl domain and triggers activation of its E3 ligase activity through an autocatalytic mechanism which amplifies its E3 ligase activity towards Mcl-1. Conclusions Autocatalytic activation of Parkin bolsters it accumulation on mitochondria and apoptotic response to valinomycin. Our results suggest that PINK1-Parkin constitutes a damage-gated molecular switch that governs cellular context-specific cell fate decisions in response to variable stress stimuli.

BACKGROUND AND PURPOSEThe toll-like receptor TLR4 is involved in neuropathic pain and in drug reward and reinforcement. The opioid inactive isomers (+)-naltrexone and (+)-naloxone act as TLR4 antagonists, reversing neuropathic pain and reducing opioid and cocaine reward and reinforcement. However, how these agents modulate TLR4 signalling is not clear. Here, we have elucidated the molecular mechanism of (+)-naltrexone and (+)-naloxone on TLR4 signalling. EXPERIMENTAL APPROACHBV-2 mouse microglial cell line, primary rat microglia and primary rat peritoneal macrophages were treated with LPS and TLR4 signalling inhibitors. Effects were measured using Western blotting, luciferase reporter assays, fluorescence microscopy and ELISA KEY RESULTS(+)-Naltrexone and (+)-naloxone were equi-potent inhibitors of the LPS-induced TLR4 downstream signalling and induction of the pro-inflammatory factors NO and TNF-α. Similarly, (+)-naltrexone or (+)-naloxone inhibited production of reactive oxygen species and increased microglial phagocytosis, induced by LPS. However, (+)-naltrexone and (+)-naloxone did not directly inhibit the increased production of IL-1β, induced by LPS. The drug interaction of (+)-naloxone and (+)-naltrexone was additive. (+)-Naltrexone or (+)-naloxone inhibited LPS-induced activation of IFN regulatory factor 3 and production of IFN-β. However, they did not inhibit TLR4 signalling via the activation of either NF-κB, p38 or JNK in these cellular models. CONCLUSIONS AND IMPLICATIONS(+)-Naltrexone and (+)-naloxone were TRIF-IFN regulatory factor 3 axis-biased TLR4 antagonists. They blocked TLR4 downstream signalling leading to NO, TNF-α and reactive oxygen species. This pattern may explain, at least in part, the in vivo therapeutic effects of (+)-naltrexone and (+)-naloxone. AbbreviationsAP-1, activator protein 1; IRF3, IFN regulatory factor 3; MD2, myeloid differentiation protein 2; MyD88, myeloid differentiation primary response 88; NBT, nitroblue tetrazolium; STAT, signal transducer and activator of transcription; TLR4, tolllike receptor 4; TRIF, TIR-domain-containing adapter-inducing IFN-β BJP British Journal of Pharmacology

Single-walled carbon nanotubes (SWNTs) binding to human telomeric i-motif DNA can significantly accelerate S1 nuclease cleavage rate by increasing the enzyme turnover number.

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib.

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly(rA)] tail at the 3'-end and human poly(rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(rA) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly(rA) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry. SWNTs have shown potential applications that range from nanodevices, gene therapy, and drug delivery to membrane separations. Our studies may provide new insights into the application of SWNTs under physiological conditions, possibly being used as probes that target specific gene sequences.