By using the aptamer proximity binding assay strategy, the development of a label-free and homogeneous approach for fluorescent detection of human platelet-derived growth factor BB (PDGF-BB) is described. Two G-quadruplex forming sequence-linked aptamers bind to the PDGF-BB proteins, which leads to the increase in local concentration of the aptamers and promotes the formation of the G-quadruplex structures. Subsequently, the fluorescent dye, N-methylmesoporphyrin IX, binds to these G-quadruplex structures and generates enhanced fluorescence emission signal for sensitive detection of PDGF-BB. The association of the aptamers to the PDGF-BB proteins is characterized by using native polyacrylamide gel electrophoresis. The experimental conditions are optimized to reach an estimated detection limit of 3.2nM for PDGF-BB. The developed method is also selective and can be applied for monitoring PDGF-BB in human serum samples. With the advantages of label-free and homogeneous detection, the demonstrated approach can be potentially employed to detect other biomarkers in a relatively simple way.

建立了液相色谱-串联质谱(HPLC-MS/MS)法同时检测浓缩樱桃李汁中罗丹明B和苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ5种染料的分析方法.样品经水稀释、乙腈提取,凝胶渗透色谱(GPC)净化,采用Zorbax Eclipse Plus C18色谱柱进行分离.在电喷雾正离子模式下(ESI+),用多重反应监测(MRM)方式进行离子监测,基质匹配外标法定量.5种染料在各自线性范围内,相关系数(r2)均大于0.998,检出限(信噪比=3)为0.2～3.0μg/kg,定量限(信噪比=10)为0.5～10.0 μg/kg,回收率在74.4％～95.3％之间,相对标准偏差为3.6％～7.9％.

The organic cation transporter (OCT, SLC22) family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs, toxins, and endogenous metabolites in a variety of tissues. Many of the OCTs have been previously characterized, but there are a number of orphan genes whose functions remain unknown. In this study, two novel rat SLC22 genes, SLC22A17 (BOCT1) and SLC22A23 (BOCT2), were cloned and characterized. Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues. BOCT1 was strongly expressed in brain, primary neurons and brain endothelial cells, with highest expression in choroid plexus. BOCT2 was also abundantly expressed in brain, as well as in liver. To characterize the products of these genes, BOCT1 cDNA was isolated from a rat blood–brain barrier cDNA library, and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques. Plasmids expressing BOCT1 and BOCT2 were transfected into HEK-293 cells, as were control cDNAs for OCT1 and OCTN2. Recombinant cell surface protein was verified by western blot and fluorescence microscopy. Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays. The OCT1- and OCTN2-expressing cells transported the canonical substrates, 1-methyl-4-phenyl-pyridinium (MPP+) and carnitine, respectively. However, BOCT1 and BOCT2-expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates. These novel family members have a nonconserved amino terminus, relative to other OCTs, that may preclude typical SLC22 transport function.

Contamination by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been identified at areas of explosive manufacturing, processing, storage, and usage. Anaerobic conversion of RDX to N-nitroso metabolites (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX)) has been demonstrated in the environment and in gastrointestinal tracts of mammals in vivo. Thus, potential exists for exposure to these N-nitroso compounds. While exposed to TNX via drinking water ad libitum, deer mice (Peromyscus maniculatus) were bred in three generations to produce cohorts F1A-D, F2A-B, and F3A. TNX was administered at four exposure levels: control (0 microg L(-1)), 10 microg L(-1), 100 microg L(-1), and 1000 microg L(-1). Endpoints investigated include: offspring production, offspring survival, offspring weight gain, and offspring organ weights. TNX exposure decreased litter size and increased postpartum mortality of offspring at the highest exposure level.

We evaluated the use of the gas exchange rate as an ecologically relevant indicator of chemical stress in avian embryos/eggs. Northern bobwhite quail (Colinus virginianus) were exposed to octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) via feed containing nominal concentrations of 0, 12.5, 50.0, and 125.0mgkg−1. Metabolic rates (oxygen consumption) of developing quail eggs were then measured via respirometry to examine potential effects of HMX exposure. Metabolic rates were examined on 5, 9, and 21d of incubation. Next, concentrations of HMX in embryos/eggs were determined by liquid chromatography–mass spectrometry. Mean (±SE) concentrations of HMX in eggs were 21.0±5.9, 1113±79.0, 3864±154.0, and 7426±301.1ngg−1 in control, low, medium and high dose groups, respectively. There were significant differences in oxygen consumption among the three embryo ages, however differences among the ages were not consistent among dose groups (age×dose group interaction p<0.0001). Oxygen consumption rates did not vary as a function of HMX in embryos (p=0.18). No evidence was observed for alterations of in ovo metabolic rates associated with HMX exposure.

An efficient sample extraction and cleanup method was developed for determination of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in eggs. The procedure included solvent extraction of HMX from eggs followed by cleanup using florisil and styrene-divinylbenzene (SDB) cartridges. Homogenized egg aliquots were thoroughly mixed with 10mL acetonitrile and extracted with ultrasonication for 1h. Each sample was centrifuged and all liquid was collected for cleanup. After concentration by N(2) evaporation, each extract was cleaned by florisil and SDB cartridges to remove endogenous interfering compounds. Finally, each extract was filtered through a 0.2microm PTFE membrane and stored for liquid chromatographic-mass spectrometric (LC-MS) analysis. Chromatographic separation was achieved on a reverse phase (RP) C18 column, with a mobile phase containing 60% methanol+40% 1.0mM acetic acid aqueous solution. Acetic acid was employed as mobile phase additive to form negatively charged adduct ions [M+CH(3)COO](-), and m/z=355 was quantified by selective ion monitoring (SIM). Overall recoveries from eggs containing 10, 50, 250 and 1000ng/g of HMX were 84.0%, 88.0%, 90.6% and 87.4%. A method detection limit (MDL) of 0.15ng/g was achieved.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transforms anaerobically into N-nitroso compounds: hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Exposure to these N-nitroso metabolites may occur in areas contaminated with explosives, as anaerobic degradation occurs via some bacteria and is one remediation strategy used for RDX. Few papers report acute oral toxicity and none have evaluated age dependent toxicity of RDX or its N-nitroso metabolites. Median lethal dose (LD50) was determined in deer mice (Peromyscus maniculatus) of three age classifications 21 d, 50 d, and 200 d for RDX, MNX, and TNX using the US EPA up-and-down procedure (UDP). Hexahydro-1,3,5-trinitro-1,3,5-triazine and N-nitroso metabolites caused similar overt signs of toxicity. Median lethal dose for 21 d deer mice were 136, 181, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 50 d deer mice were 319, 575, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 200 d deer mice were 158, 542, and 999 mg/kg for RDX, MNX, and TNX, respectively. These data suggest that RDX is the most potent compound tested, and age dependent toxicity may exist for all compounds and could play a role in RDX and RDX N-nitroso metabolite ecological risk evaluation of terrestrial wildlife at RDX contaminated sites.

Adult Northern bobwhite quail (Colinus virginianus) were exposed via food to octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), an energetic compound found in soils at military training installations. Depuration of HMX into eggs was examined in an initial study, and effects on egg production, hatching, growth, development, and survival of chicks were examined in a follow-up study. HMX was readily and rapidly transferred from female quail into eggs. Marked weight loss was observed in quail exposed to 125 and 250 mg/kg HMX in food, likely due to reductions in food intake rather than a toxic mechanism. In the second study, significant alterations in body mass occurred among quail at concentrations >52.5 +/- 9.3 mg/kg but not at 12.3 +/- 1.1 mg/kg in food. Treatment-related reductions in food consumption and decreases in egg laying rates were observed. No HMX-related effects were found in chick growth or survival. Quail inhabiting HMX-contaminated sites could possibly be exposed to HMX and therefore deposition of HMX into eggs is also possible. However, results of these studies further suggest that the potential for reproductive toxicity of HMX to birds is low.

Runoff of organic and inorganic contaminants from live firing ranges is a challenging issue because of the extensive size, the variable nature of runoff, the random occurrence of surface contamination, and general inaccessibility. This is particularly true with energetic compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). One potentially promising technology for addressing runoff in a passive, sustainable, and low-cost manner is the use of constructed wetlands to intercept flow. This research effort investigated the ability of down-flow constructed wetlands to remediate RDX-contaminated water at varying loading rates over a period of 2 years. The impact of competing electron acceptors (NO3− and SO42−), the role of plants in the remediation process, and the production of initial daughter products were determined. Significant RDX removal occurred (89–96%) for all loading rates (160–1600mg/(m2day)) at a hydraulic retention time of approximately 2 days. RDX degradation occurred in both NO3− and SO42− dominated electron acceptor zones. RDX and NO3− degradation rate constants (kRDX and kNO3−) were also determined using a 1D transport model with dispersion. KRDX declined with the increase of influent RDX (6.2−0.8d−1 for planted mesocosms). Changes in degradation rates may be the result of changing bioavailability of carbon released by the peat moss. kNO3− followed similar trends but was relatively greater than kRDX. Removal of plants is believed to have a small impact on overall RDX removal. RDX concentration in plant tissue was found to be semi-logarithmically related to RDX concentration in the rhizosphere. MNX, DNX, and TNX were observed as transient products generally proportional to RDX loading and low concentrations of MNX and DNX were detected in plant tissue at high RDX loading rates. Cessation of RDX exposure substantially reduced RDX concentrations plants over 6 weeks. These results support the use of constructed wetlands for the remediation of low-level RDX-contaminated water.

A quantitative liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the analysis of the explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). In negative ionization mode, HMX forms an acetate adduct ion [M + CH(3)COO](-), m/z 355, in the presence of a small amount of acetic acid in the mobile phase. The ESI collision-induced dissociation (CID) spectrum of m/z 355 was acquired and the transitions m/z 355 --> 147 and m/z 355 --> 174 were chosen for the determination of HMX in samples. Using this quantification technique, the method detection limit was 1.57 microg/L and good linearity was achieved in the range 5-500 microg/L. This method will help to unambiguously analyze environmentally relevant concentrations of HMX.