Purified fluorescent nanohybrids based on quantum dot-HER2-antibody for breast tumor target imaging.

Quantum dots (QDs) have been widely used for bioimaging in vivo because of their excellent optical properties. As part of the preparation process of QD-based nanohybrids, purification is an important step for minimizing contaminants and improving the quality of the product. In this work, we describe high-performance size exclusion chromatography (HPSEC) used to purify nanohybrids of CdSe/ZnS QDs and anti-human epidermal growth factor receptor 2 antibodies (QD-HER2-Ab). The unbound antibody and suspended agglomerates were removed from freshly prepared QD-HER2-Ab via HPSEC. Pure and homogeneous QD-HER2-Ab were then used as immunofluorescence target imaging bioprobes in vivo. The QD-HER2-Ab did not cause any obvious acute toxicity in mice one week after a single intravenous injection of 15 nmol/kg. The purified QD-HER2-Ab bioprobes showed high tumor targeting ability in a human breast tumor xenograft nude mouse model (24 h after injected) with the possibility of in vivo immunofluorescence tumor imaging. The immunofluorescence imaging background signal and acute toxicity in vivo were minimized because of the reduction of residual QDs. HPSEC-purified QD-HER2-Ab is an accurate and convenient tool for in vivo tumor target imaging and HER2 detection, thus providing a basis for the purification of other QD-based bioprobes.

Quaternary ammonium iminofullerenes improve root growth of oxidative-stress maize through ASA-GSH cycle modulating redox homeostasis of roots and ROS-mediated root-hair elongation.

BACKGROUND: Various environmental factors are capable of oxidative stress to result in limiting plant development and agricultural production. Fullerene-based carbon nanomaterials can enable radical scavenging and positively regulate plant growth. Even so, to date, our knowledge about the mechanism of fullerene-based carbon nanomaterials on plant growth and response to oxidative stress is still unclear. RESULTS: 20 or 50 mg/L quaternary ammonium iminofullerenes (IFQA) rescued the reduction in root lengths and root-hair densities and lengths of Arabidopsis and maize induced by accumulation of endogenous hydrogen peroxide (H2O2) under 3-amino-1,2,4-triazole or exogenous H2O2 treatment, as well as the root active absorption area and root activity under exogenous H2O2 treatment. Meanwhile, the downregulated contents of ascorbate acid (ASA) and glutathione (GSH) and the upregulated contents of dehydroascorbic acid (DHA), oxidized glutathione (GSSG), malondialdehyde (MDA), and H2O2 indicated that the exogenous H2O2 treatment induced oxidative stress of maize. Nonetheless, application of IFQA can increase the ratios of ASA/DHA and GSH/GSSG, as well as the activities of glutathione reductase, and ascorbate peroxidase, and decrease the contents of H2O2 and MDA. Moreover, the root lengths were inhibited by buthionine sulfoximine, a specific inhibitor of GSH biosynthesis, and subsequently rescued after addition of IFQA. The results suggested that IFQA could alleviate exogenous-H2O2-induced oxidative stress on maize by regulating the ASA-GSH cycle. Furthermore, IFQA reduced the excess accumulation of ROS in root hairs, as well as the NADPH oxidase activity under H2O2 treatment. The transcript levels of genes affecting ROS-mediated root-hair development, such as RBOH B, RBOH C, PFT1, and PRX59, were significantly induced by H2O2 treatment and then decreased after addition of IFQA. CONCLUSION: The positive effect of fullerene-based carbon nanomaterials on maize-root-hair growth under the induced oxidative stress was discovered. Application IFQA can ameliorate oxidative stress to promote maize-root growth through decreasing NADPH-oxidase activity, improving the scavenging of ROS by ASA-GSH cycle, and regulating the expressions of genes affecting maize-root-hair development. It will enrich more understanding the actual mechanism of fullerene-based nanoelicitors responsible for plant growth promotion and protection from oxidative stress.

Purification of quantum dot-based bioprobes via high-performance size exclusion chromatography.

Due to excellent optical properties, quantum dots (QDs) have been widely applied to sensing, labeling, and imaging. For the fabrication of QD-based bioprobes, purification is usually the crucial step. Hydrophilic octylamine grafted polyacrylic acid modified QDs (OPA-QDs) were prepared, and purified by high-performance size exclusion chromatography (HPSEC) to remove excess OPA and aggregated QDs. The percentage of suspended agglomerates of OPA-QDs in the unpurified OPA-QDs increases from 4% to 31% through a year, but the purified OPA-QDs of the same batch possess excellent colloidal stability for at least one year. Subsequently, QD-based bioprobes were fabricated by the conjugation between QDs and streptavidin (SA) or antibody (IgG), generating QD-SA and QD-IgG, respectively, which were purified via HPSEC. Finally, the resulting QD-SA and QD-IgG were adopted to detect tumour markers on slices and showed specific positive signals without nonspecific adsorption, which was contrary to the unpurified QD-IgG. Thus, the HPSEC-coupled system proposed in the current work is potent and universal for the generation of purified and monodisperse QD-based bioprobes, which is promising in the nanobiodetection field.

In vitro modeling of COPD inflammation and limitation of p38 inhibitor - SB203580.

BACKGROUND: Systemic inflammation and steroid resistance are the hallmarks of COPD. We examined the impact of p38 inhibitor (SB203580) in in vitro assays of systemic inflammation using pulmonary cells and patients' sera. OBJECTIVE AND METHODS: Data from 66 COPD patients and 15 age-/sex-matched healthy controls were compared. Interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and CCL5 were measured in serum samples and culture media from peripheral blood mononuclear cells. The impact of sera on IL-10 and CCL5 expression in alveolar macrophage cell line (MH-S) was examined. The in vitro effects of SB203580 on lipopolysaccharide-induced inflammation were investigated. RESULTS: Peripheral blood mononuclear cells from Global initiative for chronic Obstructive Lung Disease (GOLD) D patients produced more CCL5 and TNF-α, and less IL-10 compared to GOLD A-C patients. SB203580 treatment suppressed CCL5 and TNF-α and stimulated IL-10 production; however, the effect of SB203580 on IL-10 was lower in the COPD group. Culture of MH-S cells with COPD serum showed a significant increase in CCL5 and a significant decrease in IL-10 compared to healthy serum. This effect was not suppressed with SB203580 treatment. CONCLUSION: COPD serum has a potent proinflammatory effect on pulmonary cells. Inhibition of p38 phoshorylation had a limited effect in restoring impaired lymphocyte function and suppressing inflammation induced by COPD serum, implying important p38-independent inflammatory mechanisms in COPD.

Additive Suppression of LPS-Induced IL-10 and TNF-α by Pre-treatment of Dexamethasone and SB203580 in a Murine Alveolar Macrophage Cell Line (MH-S).

P38 inhibitors are potent anti-inflammatory agents with distinctive mechanism of action from corticosteroid; the potential combined use of both anti-inflammatory agents could be an effective treatment for inflammatory lung disease; however, the impact of such combination on the homeostasis of immune response was poorly understood. To investigate the combined effect of dexamethasone (DEX) and/or SB203580 on tumor necrosis factor (TNF)-α (pro-inflammatory) and interleukin (IL)-10 (anti-inflammatory) secretion in mouse alveolar macrophage cell line. Secreted TNF-α and IL-10 were measured by ELISA. Phosphorylated STAT3 were investigated using Western blotting and immunocytochemistry. Pre-treatment of DEX or SB203580 inhibited lipopolysaccharide (LPS)-stimulated IL-10, TNF-α secretion, and STAT3 phosphorylation. Combined use of both agents showed stronger inhibitory effect. Combining DEX and SB203580 showed strong inhibition on the LPS-induced IL-10 secretion and STAT3 phosphorylation, which might reflect a very important drawback from the combined use of both anti-inflammatory agents.

Interaction between glutathione S-transferase variants, maternal smoking and childhood wheezing changes with age.

BACKGROUND: Maternal smoking increases the risk of respiratory symptoms in children. Glutathione S-transferases (GSTs) detoxify xenobiotics from tobacco smoke, and functional polymorphism in GST gene(s) could predispose children to the detrimental effects of maternal smoking. Our objective was to investigate interactions between GST variants and maternal smoking in relation to the development of wheezing during childhood and whether any such interaction changes with time. METHODS: In a population-based birth cohort, we assessed maternal smoking and current wheeze at five time points during the first 11 yr of life. DNA was genotyped for GSTP1, GSTM1 and GSTT1 (n = 807). Longitudinal analyses were performed using generalized estimating equations. RESULTS: During early childhood, children whose mothers smoked were more likely to wheeze, with the strongest association observed at age 3 yr (p = 0.006). In a longitudinal model, children with GSTP1 AA and AG genotypes had significantly higher risk of wheeze compared with GG homozygotes. We observed a significant interaction between GSTP1 and maternal smoking where the risk of infantile wheezing was significantly increased in AA homozygotes, but only if their mothers smoked (OR 2.59, [1.08-6.21], p(int) = 0.03). Furthermore, amongst AA carriers, there was a significant interaction between child's age and maternal smoking, with the effect of maternal smoking on the risk of wheeze significantly diminishing with age (p(int) = 0.05); no such findings were observed for GSTM1 and GSTT1. CONCLUSIONS: Children with AA genotype for GSTP1 are at increased risk of early-life wheezing if their mothers smoke, but the effect of maternal smoking on wheezing diminishes with time.

Genetic variation in vascular endothelial growth factor-a and lung function.

RATIONALE: Given the role of vascular endothelial growth factor (VEGF) in lung development, we hypothesized that polymorphisms in VEGF-A may be associated with lung function. OBJECTIVES: The current study was designed to assess the role of genetic variants in VEGF-A as determinants of airway function from infancy through early adulthood. METHODS: Association between five single-nucleotide polymorphisms (SNPs) in VEGF-A and lung function were assessed longitudinally in two unselected birth cohorts and cross-sectionally among infants. Replication with two SNPs was conducted in adults and children with asthma. We investigated the functionality of the SNP most consistently associated with lung function (rs3025028) using Western blotting to measure the ratio of plasma VEGF-A(165b)/panVEGF-A(165) among homozygotes. MEASUREMENTS AND MAIN RESULTS: In two populations in infancy, C-allele homozygotes of rs3025028 had significantly higher VmaxFRC, forced expiratory flow(50), and forced expiratory flow(25-75) compared with other genotype groups. Among preschool children (age 3 yr), C allele of rs3025028 was associated with significantly higher specific airway conductance, with similar findings observed for lung function in school-age children. For FEV(1)/FVC ratio similar findings were observed among adolescents and young adults (birth cohort), and then replicated in adults and schoolchildren with asthma (cross-sectional studies). For rs3025038, plasma VEGF-A(165b)/panVEGF-A(165) was significantly higher among CC versus GG homozygotes (P ≤ 0.02) at birth, in school-age children, and in adults. CONCLUSIONS: We report significant associations between VEGF-A SNP rs3025028 and parameters of airway function measured throughout childhood, with the effect persisting into adulthood. We propose that the mechanism may be mediated through the ratios of active and inhibitory isoforms of VEGF-A(165), which may be determined by alternative splicing.