Assembled Step Emulsification Device for Multiplex Droplet Digital Polymerase Chain Reaction.

Digital PCR is a powerful method for absolute nucleic acid quantification with unprecedented accuracy and precision. To promote the wider use and application of digital PCR, several major challenges still exist, including reduction of cost, integration of the instrumental platform, and simplification of operations. This paper describes a reusable microfluidic device that generates nanoliter droplet arrays based on step emulsification for the on-chip multiplex digital PCR of eight samples simultaneously. The device contains two glass plates that can be quickly assembled with prefilled mineral oil. Droplets are simply generated through the arrays of step emulsification nozzles driven by a single pressure controller and are self-assembled into monolayer droplet arrays in U-shaped chambers. The use of mineral oil eliminates bubble generation; thus, no overpressure is required during thermocycling. Moreover, the device can be reused many times after disassembly and a brief cleaning procedure, which significantly reduces the cost of the device per dPCR assays. The device was able to detect template DNA at concentrations as low as 10 copies/µL with a dynamic range of approximately 4 logs. We applied this device in the quantitative assessment of HER2 copy number variation, which is important for targeted therapy and prognosis of breast cancer. The performance was validated by 16 clinical samples, obtaining similar results to commercial digital PCR. We envision that this low-cost, reusable, and user-friendly device can be broadly used in various applications.

Mechanism of protein decarbonylation.

Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling.