Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

Effects of bilateral auricular acupuncture stimulation on body weight in healthy volunteers and mildly obese patients.

We investigated the effects of auricular acupuncture stimulation on non-obese healthy volunteers and mildly obese patients. Subjects (n = 55 and 5, respectively) averaged 34.5 years old, and BMI was 24.3 and less than 27.5 kg/m2, respectively. We also studied the effects of single-blind sham treatment in approximately 500 age-, sex-, and BMI-matched subjects. Small (0.15 x 2.0 mm) auricular needles were placed intracutaneously into the bilateral cavum conchae identified by having a resistance of less than 100 kOmega/cm2. In the 2-week pretreatment the period, in which body weight was measured without auricular acupuncture stimulation, 57.1% of the subjects showed a reduction in body weight. This indicates that charting one's own body weight might itself be a useful method of weight control. In the auricular acupuncture treatment period, 35 healthy subjects of 55 (63.6%) showed a decreased body weight, 11 (20%) showed an increased body weight, and 9 (16.4%) showed no change in body weight. The obese patients showed individual variation, but all achieved weight reduction, with a highly significant correlation between body weight and fat volume. The CT/MRI cross-sectional pictures supported these findings. Sham treatment had no statistically significant effect on body weight. These results suggest that success in achieving weight reduction can be partly attributed to the act of charting of one's own weight pattern. Bilateral auricular acupuncture stimulation can help reduce body weight both in mildly obese patients and in healthy non-obese subjects. In conclusion, this is in accord with the bilateral auricular acupuncture stimulation that it may be useful in the treatment of the obesity. We propose a possible mechanism for the weight-reducing effects of bilateral auricular acupuncture stimulation.