Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC‐MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5–4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross‐reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC‐MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC‐MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents

In this report, we present a breast imaging technique combining high‐resolution near‐infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye‐labeled amino‐terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830‐ATF‐IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830‐ATF‐IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4‐ and 10‐fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non‐targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor‐targeted NIR830‐ATF‐IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization

Background

As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model.

Methods and Results

C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5–8 and 16–20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16–20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14^th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other.

Conclusion

The current results suggest that 16–20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14^th day after photocoagulation.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.     

Fluoride and Arsenic Exposure Impairs Learning and Memory and Decreases mGluR5 Expression in the Hippocampus and Cortex in Rats

Fluoride and arsenic are two common inorganic contaminants in drinking water that are associated with impairment in child development and retarded intelligence. The present study was conducted to explore the effects on spatial learning, memory, glutamate levels, and group I metabotropic glutamate receptors (mGluRs) expression in the hippocampus and cortex after subchronic exposure to fluoride, arsenic, and a fluoride and arsenic combination in rats. Weaned male Sprague-Dawley rats were assigned to four groups. The control rats drank tap water. Rats in the three exposure groups drank water with sodium fluoride (120 mg/L), sodium arsenite (70 mg/L), and a sodium fluoride (120 mg/L) and sodium arsenite (70 mg/L) combination for 3 months. Spatial learning and memory was measured in Morris water maze. mGluR1 and mGluR5 mRNA and protein expression in the hippocampus and cortex was detected using RT-PCR and Western blot, respectively. Compared with controls, learning and memory ability declined in rats that were exposed to fluoride and arsenic both alone and combined. Combined fluoride and arsenic exposure did not have a more pronounced effect on spatial learning and memory compared with arsenic and fluoride exposure alone. Compared with controls, glutamate levels decreased in the hippocampus and cortex of rats exposed to fluoride and combined fluoride and arsenic, and in cortex of arsenic-exposed rats. mGluR5 mRNA and protein expressions in the hippocampus and mGluR5 protein expression in the cortex decreased in rats exposed to arsenic alone. Interestingly, compared with fluoride and arsenic exposure alone, fluoride and arsenic combination decreased mGluR5 mRNA expression in the cortex and protein expression in the hippocampus, suggesting a synergistic effect of fluoride and arsenic. These data indicate that fluoride and arsenic, either alone or combined, can decrease learning and memory ability in rats. The mechanism may be associated with changes of glutamate level and mGluR5 expression in cortex and hippocampus.     

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

Aerobic Exercise Inhibits Sympathetic Nerve Sprouting and Restores β-Adrenergic Receptor Balance in Rats with Myocardial Infarction

Background

Cardiac sympathetic nerve sprouting and the dysregulation of β-adrenergic receptor (β-AR) play a critical role in the deterioration of cardiac function after myocardial infarction (MI). Growing evidence indicates that exercise provides protection against MI. The aims of this study were to investigate whether aerobic exercise following MI could inhibit sympathetic nerve sprouting and restore the balance of β3-AR/β1-AR.

Methods

Male Sprague-Dawley rats were divided into three groups: sham-operated control group (SC), MI group (MI), and MI with aerobic exercise group (ME). The rats in ME group were assigned to 8 weeks of exercise protocol (16 m/min, 50 min/d, 5 d/wk). The expression of nerve growth factor (NGF), the sympathetic nerve marker-tyrosine hydroxylase (TH), the nerve sprouting marker-growth associated protein 43 (GAP43), and β1- and β2-AR expression in the peri-infarct area of the left ventricle (LV) were measured by Western blot and immunohistochemistry, while β3-AR expression was determined by Western blot and immunofluorescence. Endothelial nitric oxide synthase (NOS2), phospho-NOS2 (p-NOS2), and neuronal nitric oxide synthase (NOS1) were measured by Western blot.

Results

MI increased LV end-diastolic pressure (LVEDP), and decreased LV systolic pressure (LVSP). Compared with the MI group, aerobic exercise significantly decreased LVEDP and increased LVSP. The protein expression of TH, GAP43 and NGF was significantly increased after MI, which was normalized by exercise. Compared with the SC group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were elevated in the MI group, and the protein expression of β3-AR and NOS1 increased after MI. Compared with the MI group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were normalized in the ME group, while the protein expression of β3-AR and NOS1 significantly increased, and NOS2 was activated by exercise.

Conclusions

Aerobic exercise inhibits cardiac sympathetic nerve sprouting, restores β3-AR/β1-AR balance and increases β3-AR expression through the activation of NOS2 and NOS1 after myocardial infarction.     

Rhegmatogenous Retinal Detachment Surgery in Elderly People over 70 Years Old: Visual Acuity,Quality of Life,and Cost-Utility Values

Background and Purpose

To evaluate the influence of rhegmatogenous retinal detachment (RRD) surgery on elderly patients in terms of visual acuity, vision-related quality of life and its cost-effectiveness.

Methods

Elderly patients over 70 years old, who were diagnosed and underwent RRD surgery at Shanghai First People''s Hospital, Shanghai Jiao Tong University, China, from January 1, 2009, through January 1, 2013. The participants received scleral buckling surgery and vitreous surgery with or without scleral buckling under retrobulbar anesthesia. We followed the patients for 1 year and collected best-corrected visual acuity (BCVA), vision-related quality of life, and direct medical costs data. Utility values elicited by time-trade-off were analyzed to determine the quality of life. Quality-adjusted life years (QALYs) gained in life expectancy were calculated and discounted at 3% annually. Costs per QALY gained were reported using the bootstrap method. Further analyses were made for two age groups, age 70–79 and age over 80 years. Sensitivity analyses were performed to test stability of the results.

Results

98 patients were included in the study. The BCVA significantly improved by 0.53±0.44 (Logarithm of the Minimum Angle of Resolution (logMAR)) at the 1-year postoperative time point (p<0.001). Utility values increased from 0.77 to 0.84 (p<0.001), and an average of 0.4 QALYs were gained in the life expectancy. Costs per QALY gained from the RRD surgery were 33,186 Chinese Yuan (CNY) (5,276 US dollars (USD))/QALY; 24,535 CNY (3,901 USD)/QALY for the age group of 70–79 years and 71,240 CNY (11,326 USD)/QALY for the age group over 80 years.

Conclusions

RRD surgery improved the visual acuity and quality of life in the elderly patients over 70 years old. According to the World Health Organization’s recommendation, at a threshold of willingness to pay of 115,062 CNY (18,293 USD)/QALY, RRD surgery is cost effective in the elderly patients.