Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

Antibodies are host proteins that comprise one of the principal effectors of the adaptive immune system. Their utility has been harnessed as they have been and continue to be used extensively as a diagnostic and research reagent. They are also becoming an important therapeutic tool in the clinician's armamentarium to treat disease. Antibodies are utilized for analysis, purification, and enrichment, and to mediate or modulate physiological responses. This overview of the structure and function of polyclonal and monoclonal antibodies describes features that distinguish one from the other. A limited review of their use as specific research, diagnostic, and therapeutic reagents and a list of printed and electronic resources that can be utilized to garner additional information on these topics are also included.

A major challenge in the synthesis of nanotube or nanowire sensors is to impart selective analyte binding through means other than covalent linkages, which compromise electronic and optical properties. We synthesized a 3,4-diaminophenyl-functionalized dextran (DAP-dex) wrapping for single-walled carbon nanotubes (SWNTs) that imparts rapid and selective fluorescence detection of nitric oxide (NO), a messenger for biological signalling. The near-infrared (nIR) fluorescence of SWNT(DAP-dex) is immediately and directly bleached by NO, but not by other reactive nitrogen and oxygen species. This bleaching is reversible and shown to be caused by electron transfer from the top of the valence band of the SWNT to the lowest unoccupied molecular orbital of NO. The resulting optical sensor is capable of real-time and spatially resolved detection of NO produced by stimulating NO synthase in macrophage cells. We also demonstrate the potential of the optical sensor for in vivo detection of NO in a mouse model.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen͞ nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.Wilson disease ͉ hemochromatois ͉ p53 ͉ iron ͉ copper ͉ liver carcinogenesis

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosomedependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death. signal transduction | cancer therapyA utophagy is a self-digestion process by which a eukaryotic cell degrades and recycles aggregate-prone proteins, macromolecules, and organelles. During autophagy, cytoplasmic contents are sequestered in double-membrane bound vesicles called autophagosomes and delivered to lysosomes for degradation, thereby allowing cells to eliminate and recycle the contents (1-3). Autophagy participates in both prosurvival (recycling of cellular building blocks) and prodeath (excess catalysis) pathways. A comprehensive understanding of signaling pathways that regulate autophagy holds great promise for new therapeutic opportunities by opening the possibility to compromise prosurvival autophagic pathways that enable tumor cells to evade therapy, or by promoting prodeath autophagic pathways to kill cancer cells.The classical pathway regulating autophagy in mammalian cells involves the serine/threonine kinase, mammalian target of rapamycin (mTOR). Active mTOR kinase in the mTORC1 complex phosphorylates and inhibits ULK1, a key proautophagy adapter involved in nucleation of the autophagophore membrane. Inactivation of mTORC1, either pharmacologically with rapamycin or via activation of the tuberous sclerosis complex (TSC) tumor suppressor, leads to downstream dephosphorylation events, including loss of ULK1 phosphorylation at S757. The TSC1/2 heterodimer is itself regulated by upstream kinases, including the AMP-activated protein kinase (AMPK), which regulates several metabolic processes and activates t...

IntroductionMultiple myeloma (MM) is a B-cell malignancy characterized by proliferation of monoclonal plasma cells in the bone marrow (BM). Despite the recent emergence of novel therapies including bortezomib, 1,2 thalidomide, 3,4 and lenalidomide, 5 it remains incurable due to the development of drug resistance. [5][6][7] Among the factors that lead to this resistance are defects in apoptotic signaling pathways and overexpression of the multidrug resistance protein (MRP) pumps that enhance drug efflux. 8 In addition, the BM microenvironment confers drug resistance in MM via (1) secretion of cytokines such as interleukin 6 (IL-6) and insulin-like growth factor 1 (IGF-1), which mediate survival signals in MM cells, 9-11 as well as (2) direct interaction with MM cells, which results in cell adhesion-mediated drug resistance. 12,13 Despite recent progress, MM remains incurable, and new therapeutic agents with novel mechanism of actions are urgently needed.JS-K (O 2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a diazeniumdiolate class of prodrug designed to release nitric oxide (NO • ) when metabolized by glutathione S-transferases (GSTs; Figure 1A). 14 GSTs are enzymes that catalyze the conjugation of xenobiotics with glutathione (GSH), thereby facilitating their subsequent efflux through MRP pumps. 15 GSTs are frequently overexpressed in a broad spectrum of tumors. 16,17 In the context of conventional chemotherapy, this provides tumor cells with a selective survival advantage over normal cells by enhancing drug efflux and thus decreasing therapeutic efficacy. In contrast, since JS-K uniquely requires GST for its optimal activity, it can potentially turn GST overexpression to the tumor's disadvantage by generating relatively high intracellular concentrations of cytotoxic NO • , specifically within tumor cells. Importantly, JS-K has recently been shown to inhibit tumor growth in both in vitro and in vivo models of human prostate cancer and human leukemia. 14 Importantly, GSTs are overexpressed in 10% to 70% of patients with MM at diagnosis, and in 30% of patients at relapse. 8 In addition, in our recent study comparing gene expression profiles of patient MM cells with normal plasma cells from a genetically identical twin, we observed that GST was overexpressed by 7-fold in MM cells. 18 Furthermore, in our high-resolution genomic and expression profiling of primary tumor cells from 67 patients with MM and plasma cells from 12 healthy donors, 19 33% and 39% of the MM cells overexpressed GSTP1 and GSTM1, respectively, when compared with plasma The online version of this manuscript contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 10, 2018. by guest www.bloodjournal.org From cell controls. To date, however, the biological effects of JS-K on MM cells ha...

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Nitric oxide (NO