Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology-Based EGFR Mutation Assay in Patients with Operable Non-Small Cell Lung Cancer.

PURPOSE: Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens. MATERIALS AND METHODS: In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non-small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing-based CancerSCAN was utilized as a referee. RESULTS: The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients. CONCLUSION: The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Biological SERS-active sensor platform based on flexible silk fibroin film and gold nanoislands.

In contrast to conventional surface-enhanced Raman scattering (SERS) platforms implemented on non-biological substrates, silk fibroin has the unique advantages of long-term biosafety and controllable biodegradability for in vitro and in vivo biomedical applications, as well as flexibility and process-compatibility. In this study, a silk fibroin film was developed to fabricate a flexible SERS sensor template with nanogap-rich gold nanoislands. The proposed biological SERS platform presents fairly good enhancements in detection performance such as detection limit, sensitivity, and signal-to-noise ratio. In particular, the sensitivity improvement was by more than 10 times compared to that of the counterpart sample, and an excellent spatial reproducibility of 2.8% was achieved. In addition, the near-field calculation results were consistent with the experimental results, and the effect of surface roughness of the silk substrate was investigated in a quantitative way. It is believed that biological SERS-active sensors could provide the potential for highly sensitive, cost-effective, and easily customizable nanophotonic platforms that include new capabilities for future healthcare devices.

Combination of Porous Silk Fibroin Substrate and Gold Nanocracks as a Novel SERS Platform for a High-Sensitivity Biosensor.

Novel concepts for developing a surface-enhanced Raman scattering (SERS) sensor based on biocompatible materials offer great potential in versatile applications, including wearable and in vivo monitoring of target analytes. Here, we report a highly sensitive SERS sensor consisting of a biocompatible silk fibroin substrate with a high porosity and gold nanocracks. Our silk-based SERS detection takes advantage of strong local field enhancement in the nanoscale crack regions induced by gold nanostructures evaporated on a porous silk substrate. The SERS performance of the proposed sensor is evaluated in terms of detection limit, sensitivity, and linearity. Compared to the performance of a counterpart SERS sensor with a thin gold film, SERS results using 4-ABT analytes present that a significant improvement in the detection limit and sensitivity by more than 4 times, and a good linearity and a wide dynamic range is achieved. More interestingly, overlap is integral, and a quantitative measure of the local field enhancement is highly consistent with the experimental SERS enhancement.

Complete Biotransformation of Protopanaxadiol-Type Ginsenosides to 20- O-ß-Glucopyranosyl-20( S)-protopanaxadiol Using a Novel and Thermostable ß-Glucosidase.

The ginsenoside 20- O-ß-glucopyranosyl-20( S)-protopanaxadiol, compound K, has attracted much attention in functional food, traditional medicine, and cosmetic industries because of diverse pharmaceutical activities. The effective production of compound K from ginseng extracts has been required. However, an enzyme capable of completely converting all protopanaxadiol (PPD)-type ginsenosides to compound K has not been reported until now. In this study, unlike other enzymes, ß-glucosidase from Caldicellulosiruptor bescii was able to hydrolyze sugar moieties such as l-arabinofuranose as well as d-glucose and l-arabinopyranose as the C-20 outer sugar in ginsenosides. Thus, ginsenoside Rc containing l-arabinofuranose can be converted to compound K by only this enzyme. Under the optimized reaction conditions, the enzyme completely converted PPD-type ginsenosides in ginseng extracts to compound K with the highest productivity among the reported results. This is the first report of the enzyme capable of completely converting all PPD-type ginsenosides into compound K.

An L213A variant of ß-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. ß-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. ß-Glycosidase from S. solfataricus can hydrolyze ß-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to ß-glycosidase from S. solfataricus in homology model and sequence was aligned with ß-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant ß-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant ß-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type ß-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

Characterization of L-rhamnose isomerase from Clostridium stercorarium and its application to the production of D-allose from D-allulose (D-psicose).

OBJECTIVE: To characterize L-rhamnose isomerase (L-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce D-allose from D-allulose. RESULTS: A recombinant L-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k cat/K m) for L-rhamnose among the reported L-RIs. The L-RI was applied to the high-level production of D-allose from D-allulose. The isomerization activity for D-allulose was maximal at pH 7, 75 °C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the L-RI at 65, 70, 75, and 80 °C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 °C. Under the optimized conditions of pH 7, 70 °C, 1 mM Mn2+, 27 U L-RI l-1, and 600 g D-allulose l-1, L-RI from C. stercorarium produced 199 g D-allose l-1 without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l-1 h-1. CONCLUSION: To the best of our knowledge, this is the highest concentration and productivity of D-allose reported thus far.

Synergistic production of 20(S)-protopanaxadiol from protopanaxadiol-type ginsenosides by ß-glycosidases from Dictyoglomus turgidum and Caldicellulosiruptor bescii.

20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because ß-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. ß-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-L-arabinopyranoside moiety in compound Y. Therefore, ß-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-L-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of ß-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far.

Engineered Salmonella typhimurium expressing E7 fusion protein, derived from human papillomavirus, inhibits tumor growth in cervical tumor-bearing mice.

The tumor-suppressing effects of SipB160/HPV16 E7 fusion protein, derived from human papillomavirus, and expressed in Salmonella enterica serovar typhimurium, were evaluated in a cervical cancer model. The expressed E7 protein resulted in efficacious cytotoxicity and tumor growth retardation in TC-1 cervical cancer cells. In addition, in mice bearing TC-1 tumors, live cells of Salmonella expressing HPV16 E7 were administered orally and induced immune responses through interferon-gamma and tumor necrosis factor-alpha cytokine secretion and also suppressed tumor growth (45 %) and prolonged survival (70 %) compared with the control group. These results suggested that the SipB160/HPV16 E7 fusion protein may be a candidate cancer therapeutic agent.

Bacteria-based in vivo peptide library screening using biopanning approach.

Traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. Due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. However, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. To overcome this inherent limitation, we have developed an in vivo peptide library screening system that allows for the identification of dissociative inhibitors of protein interactions of interest. The screening is based on the reconstitution of the cI repressor from bacteriophage lambda with high-density expression peptide library and is entirely performed in bacteria cells. Furthermore, to enhance the efficacy and sensitivity of the screening, a multiple-round biopanning approach was employed for amplification and enrichment of positive peptides. Overall, this in vivo screening should provide a fast and efficient tool for identification of biologically active peptide molecules against target protein assembly.