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.

Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.

Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signaling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and helper NLR activation of defense requires the lipase-domain proteins EDS1, SAG101, and PAD4. Previously, we found that NRG1 associates with EDS1 and SAG101 in a TNL activation-dependent manner [X. Sun et al., Nat. Commun. 12, 3335 (2021)]. We report here how the helper NLR NRG1 associates with itself and with EDS1 and SAG101 during TNL-initiated immunity. Full immunity requires coactivation and mutual potentiation of cell-surface and intracellular immune receptor-initiated signaling [B. P. M. Ngou, H.-K. Ahn, P. Ding, J. D. G. Jones, Nature 592, 110-115 (2021), M. Yuan et al., Nature 592, 105-109 (2021)]. We find that while activation of TNLs is sufficient to promote NRG1-EDS1-SAG101 interaction, the formation of an oligomeric NRG1-EDS1-SAG101 resistosome requires the additional coactivation of cell-surface receptor-initiated defense. These data suggest that NRG1-EDS1-SAG101 resistosome formation in vivo is part of the mechanism that links intracellular and cell-surface receptor signaling pathways.

Experiences and Perceptions of Gender Discrimination and Equality among Korean Surgeons: Results of a Survey of the Korean Surgical Society.

BACKGROUND: Previous studies that have focused on the challenges faced by female surgeons, such as the gender pay gap, gender biases, lower likelihood of promotion, and gender differences in the perception of discrimination against women, are reviewed. A more comprehensive understanding of explicit and implicit gender discrimination and experiences and perceptions of discrimination is needed. This study aims to determine the current prevalence and degree of gender discrimination in the Korean Surgical Society and to compare the experiences and perceptions of gender discrimination between male and female surgeons. METHODS: We analyzed 400 responses from a survey sent to all members of the Korean Surgical Society. This electronic survey included 16 items on experiences of gender discrimination and 17 items on perceptions of gender discrimination. We conducted χ² tests and binary logistic regression on the data regarding these experiences and perceptions of gender discrimination. RESULTS: Adjusted analyses found that female surgeons were more likely to experience gender discrimination than their male counterparts across all categories of discrimination. Further, adjusted analyses showed that female surgeons were more likely to confirm the presence of gender discrimination than male surgeons. CONCLUSION: Female surgeons were more likely to experience implicit and explicit gender biases and discrimination throughout all stages of their career progression. We also discovered significant gender differences in the perception of gender discrimination, as well as the experience of it. Changing the male-dominated culture and raising awareness of gender biases and discrimination among male surgeons are crucial steps toward addressing gender discrimination in surgery.

Profile Analysis of Sexual Assault Experiences among Adult Women and Their Implications for Mental Health.

OBJECTIVE: Extant studies indicate that just one characteristic of sexual assault cannot properly represent the whole experience of sexual assault and, especially, the severity of sexual assault. This study aimed to understand the totality of sexual assault experiences and elucidate subtypes of sexual assault victims based on the detailed characteristics of their sexual assault experiences and those relationships with mental health. METHODS: A total of 255 adult sexual violence victims who used intervention services and a comparison group were included. Information on their sexual assault experiences was gleaned from case records data. RESULTS: The following four distinctive profile groups were identified: "Sexual Touching" (19.6%), "Rape/Social Relation" (30.4%), "Intimate Partner Violence (IPV)" (18.8%), and "Rape/Stranger" (31.3%). The subgroups differed in terms of secondary victimization and adverse childhood experiences. The Rape/Social Relation and IPV subgroups most frequently experienced secondary victimization and childhood adversity. The four profile subgroups demonstrated different relationships with mental health outcomes, with a complicated pattern. The Rap/Social Relation and IPV subgroups scored higher on mental health problem screening measures compared to other groups. However, a considerable proportion of victims in the Sexual Touching subgroup also reported suicidal behaviors and self-injury. CONCLUSION: Based on the results, theoretical and clinical implications were discussed.

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.

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.