Development of a quartz crystal microbalance-based immunosensor for the early detection of mesothelin in cancer

A robust real-time flow injection assay using Quartz Crystal Microbalance (QCM) biosensor has been developed for the detection of mesothelin, an antigen expressed on various malignant tumors including mesothelioma and ovarian cancers. A QCM sensor chip functionalized with self-assembled monolayer of cysteamine used to fabricate a sensitive immunosensor. Mesothelin specific antibody was immobilized on cysteamine modified gold surface of quartz crystal using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide coupling. Further, ethanolamine was used as a blocking reagent to prevent the non-specific adsorption on antibody functionalized gold surface. Various concentrations of mesothelin was tested and the resonant frequency variation of the crystal was observed by a quartz crystal microbalance until a stable response is obtained. A good correlation between the frequency changes and concentration of mesothelin tested was observed and the QCM biosensor detects mesothelin in the linear range of 100pg/mLto 50 ng/mL. Thus, QCM assay could be a promising technique for early diagnosis of mesothelioma, ovarian and pancreatic adenocarcinoma

Photoactivable Glycolipid Antigens Generate Stable Conjugates with CD1d for Invariant Natural Killer T Cell Activation

Activation of invariant natural killer T lymphocytes (iNKT cells) by α-galactosylceramide (α-GC) elicits a range of pro-inflammatory or anti-inflammatory immune responses. We report the synthesis and characterization of a series of α-GC analogues with acyl chains of varying length and a terminal benzophenone. These bound efficiently to the glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable CD1d-glycolipid covalent conjugates. Conjugates of benzophenone α-GCs with soluble or cell-bound CD1d proteins retained potent iNKT cell activating properties, with biologic effects that were modulated by acyl chain length and the resulting affinities of conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand binding pocket of CD1d. The creation of covalent conjugates of CD1d with α-GC provides a new tool for probing the biology of glycolipid antigen presentation, as well as opportunities for developing effective immunotherapeutics

Generation and phenotypic characterisation of a cytochrome P450 4x1 knockout mouse

<div><p>Cytochrome P450 4x1 (Cyp4x1) is expressed at very high levels in the brain but the function of this protein is unknown. It has been hypothesised to regulate metabolism of fatty acids and to affect the activity of endocannabinoid signalling systems, which are known to influence appetite and energy metabolism. The objective of the present investigation was to determine the impact of Cyp4x1 on body weight and energy metabolism by developing a line of transgenic Cyp4x1-knock out mice. Mice were developed with a global knock-out of the gene; the full-length RNA was undetectable, and mice were viable and fertile. Both male and female Cyp4x1-knock out mice gained significantly more body weight on normal lab chow diet compared to control flox mice on the same genetic background. At necropsy, Cyp4x1-knock out male mice had significantly greater intra-abdominal fat deposits (P<0.01), and enlarged adipocytes. Metabolic rate and locomotor activity as inferred from VO₂ measures and crossing of infrared beams in metabolic cages were not significantly affected by the mutation in either gender. The respiratory exchange ratio was significantly decreased in male knock out mice (P<0.05), suggesting a greater degree of fat oxidation, consistent with their higher adiposity. When mice were maintained on a high fat diet, VO₂ was significantly decreased in both male and female Cyp4x1-knock out mice. We conclude that the Cyp4x1-knock out mouse strain demonstrates a mildly obese phenotype, consistent with the view that cytochrome P450 4x1 plays a role in regulating fat metabolism.</p></div

Body weight in group-housed male (Fig3A) and female (Fig3B) floxed (○, n = 7 male, n = 10 female) or Cyp4x1 knock out mice (●, n = 8 male, n = 8 female) kept on high fat diet (45% fat).

<p>Values are group mean ± SEM. Body weight for mice kept on normal lab chow (6% fat) are also depicted (flox, knock out data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187959#pone.0187959.g002" target="_blank">Fig 2</a>), note the early onset of weight gain in female knock out mice on high fat diet. Body weight *p<0.05, **p<0.01 and ***p<0.001 vs floxed controls.</p

Glucose tolerance test after high fat diet (2g/g BW, ip) in floxed (○) or Cyp4x1 knock out mice (●).

<p>Values are group mean ± SEM, group size 2–3 mice per time point.</p

Insulin tolerance test (I IU/kg BW, ip) in floxed (open bars) or Cyp4x1 knock out mice (solid bars).

<p>Values are group mean ± SEM, group size 3–4 mice per time point. **p<0.01 vs t = 0.</p

Representative epoxy-embedded sections through epididymal white adipose tissue stained with toluidine blue from a control (left) and a Cyp4x1 knock out mouse (right).

<p>Scale bar is 50 μm.</p

Total food intake (A), meal frequency (B) and meal size (C) in male (left) and female (right) floxed (open bars) or Cyp4x1 knock out mice (solid bars).

<p>Values are overall 24h mean ± SEM, n = 6 per group.</p

Cyp4x1 mRNA analysis in knock out and wild-type mouse samples.

<p>Total RNA was extracted from various tissues and reverse transcribed into cDNA. PCR was carried out using primers targeting the region between exon 3 and 5 of Cyp4x1. The upper panel shows control and knock out Cyp4x1 PCR products from different tissues and the lower panel represents amplification of GAPDH (226bps). The Cyp4x1 control samples produce an amplified product of 240 bps whereas the RNA from knock out mice results in an amplicon of 112bps.</p

Locomotor activity (A), oxygen consumption (VO₂ B), and respiratory exchange ratio (RER, C) in male (left) and female (right) floxed (○) or Cyp4x1 knock out mice (●).

<p>Grey horizontal bar depicts the dark phase. Values are hourly mean ± SEM, n = 6 per group. Overall 24h mean ± SEM values are also depicted for floxed (open bars) and Cyp4x1 knock out mice (solid bars). *p<0.05 vs floxed controls.</p