Fluorescent indicators for simultaneous reporting of all four cell cycle phases.

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

Treatment effects of microimplant-aided sliding mechanics on distal retraction of posterior teeth.

INTRODUCTION: Our objective was to quantify the treatment effects of microimplant-aided mechanics on group distal retraction of the posterior teeth. METHODS: The pretreatment and posttreatment cephalometric radiographs and dental casts of 23 patients (mean age, 22.1 ± 5.17 years), treated with distalization of the posterior teeth against microimplant anchorage and without extraction of the premolars or other teeth except the third molars, were used. The soft-tissue, skeletal, and dental measurements in the vertical and anteroposterior dimensions were analyzed. The changes in interpremolar and intermolar widths and rotations of the molars were analyzed with dental casts. RESULTS: The upper and lower lips were repositioned distally. The Frankfort horizontal to mandibular plane angle was decreased in the adult group. The maxillary posterior teeth were distalized by 1.4 to 2.0 mm with approximately 3.5° of distal tipping, and the mandibular posterior teeth were also distalized by 1.6 to 2.5 mm with approximately 6.6° to 8.3° of distal tipping. The maxillary posterior teeth showed intrusion by 1 mm. There were increases in arch widths at the premolars and molars. The overall success of microimplants was 89.7%; a well-experienced clinician had a higher success rate (98%) than did novices in this sample. The mean treatment time was 20 ± 4.9 months. CONCLUSIONS: With microimplant-aided sliding mechanics, clinicians can distalize all posterior teeth together with less distal tipping. The technique seems effective and efficient to treat patients who have mild arch length discrepancy without extractions.

Distribution of mcr genes among carbapenem-resistant Enterobacterales clinical isolates: high prevalence of mcr-positive Enterobacter cloacae complex in Seoul, Republic of Korea.

Colistin is often used as a drug of last resort against infections caused by multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacterales (CRE). Recently, the acquisition of mobile colistin resistance (mcr) genes by CRE has become a cause for concern. This study investigated the prevalence of mcr genes in CRE isolates in Seoul, Republic of Korea. In total, 3675 CRE strains were collected from patients between 2018 and 2019, and initially screened for mcr genes using multiplex polymerase chain reaction assays. Upon the identification of mcr-harbouring strains, colistin susceptibility tests, identification of carbapenemase and ß-lactamase genes, and plasmid replicon typing were performed. Clonal analysis was conducted using pulsed-field gel electrophoresis. mcr genes were detected in 2.2% (80/3675) of CRE strains. There were three mcr-1 carriers, one mcr-4.3 carrier, one mcr-4.3/mcr-9 carrier, 58 mcr-9 carriers, one mcr-9/mcr-10 carrier and 16 mcr-10 carriers among various Enterobacterales species, of which 60 were Enterobacter cloacae complex (ECC) strains. The prevalence of mcr genes in ECC strains was 20.5%. Molecular detection confirmed that 21.3% and 13.8% of mcr-harbouring strains shared blaNDM-1 or blaKPC-2, respectively. In addition, an IncHI2 replicon was identified in 71.7% of mcr-9 strains. Comparative analysis revealed not only a notable diversity of mcr carriers, but also clonal spreading or nosocomial outbreaks of some ECC strains. These findings revealed a silent distribution of mcr genes in CRE strains with high genetic heterogeneity in Seoul, underscoring the urgent need for timely intervention to control and prevent mcr dissemination.

Dissemination of an international high-risk clone of Escherichia coli ST410 co-producing NDM-5 and OXA-181 carbapenemases in Seoul, Republic of Korea.

The rapid increase in carbapenemase-producing Enterobacterales is a global health concern. During 2017-2020, a total of 44 Escherichia coli isolates co-harbouring blaNDM-5 and blaOXA-181 were collected from patients at 17 hospitals in Seoul and characterized based on antimicrobial susceptibility, resistance genes and plasmid replicons detected using polymerase chain reaction (PCR). Clonal relatedness was estimated using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates had an identical multidrug resistance profile, including resistance to carbapenems, cephalosporins, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole, and susceptibility to amikacin, colistin, and tigecycline. Resistance genes (blaCTX-M-15, blaCMY-2, blaTEM-1B, blaOXA-1, aac(6')-Ib-cr, and qnrS) and plasmid replicons (IncFIA, IncFIB, and IncX3) was observed in almost all isolates. All isolates belonged to ST410 and were genetically similar (>88% similarity), with some PFGE types shared among isolates from different hospitals. Analysis of the whole genome revealed that the isolates clustered together with other strains of the international high-risk clone ST410 B4/H24RxC from other countries. These findings underline the ongoing spread of the high-risk clone of NDM-5- and OXA-181-producing E. coli ST410 B4/H24RxC among hospitals in Seoul. Continuous monitoring and implementation of infection control measures are crucial to track and prevent further spread of these resistant strains.

Molecular characterization of partial-open reading frames 1a and 2 of the human astroviruses in South Korea.

Human astroviruses (HAstVs) are among the major causes of gastroenteritis in South Korea. In this study, the partial regions of the open reading frame (ORF) 1a and ORF2 genes of HAstVs from gastroenteritis patients in nine hospitals were sequenced, and the molecular characterization of the viruses was revealed. 89 partial nucleotide sequences of ORF1a and 88 partial nucleotide sequences of ORF2 were amplified from 120 stool specimens. Phylogenetic analysis showed that most of the nucleotide sequences of ORF1a and ORF2 were grouped with HAstV type 1 but had evolutionary genetic distance compared with the reference sequences, such as the HAstV-1 prototype, Dresden strain, and Oxford strain. According to the phylogenetic analysis, some nucleotide sequences including SE0506041, SE0506043, and SE0506058, showed the discrepancy of the genotypes, but there was no proof of recombination among the HAstV types. In conclusion, this study showed that the dominant HAstV isolated from the Seoul metropolitan area in 2004-2005 was HAstV type 1, and that Korean HAstV-1 had the genetic distance in evolution compared with the reference sequences of HAstVs. Lots of nucleotide sequences of the ORF1a and ORF2 genes of HAstV will be useful for studying for the control and prevention of HAstV gastroenteritis in South Korea.

Identification of an extensively drug-resistant Escherichia coli clinical strain harboring mcr-1 and blaNDM-1 in Korea.

The development of colistin resistance in carbapenem-resistant strains poses a serious public health problem. In this study, we collected 249 carbapenem-resistant Escherichia coli isolates from patients in Seoul in 2018, and screened all isolates for colistin resistance and for the presence of mobile colistin resistance (mcr) genes. Colistin-resistant strains were further analyzed using multilocus sequence typing, antimicrobial susceptibility testing, detection of antibiotic resistance determinants, plasmid transconjugation, and whole-genome sequencing. Three of the 249 carbapenem-resistant isolates were resistant to colistin, and mcr-1 was detected in one isolate (SECR18-0888), which belonged to sequence type 156 and was resistant to all antibiotics tested except tigecycline. The mcr-1.1 gene was located on an ~62 kb self-transferable IncI2 plasmid along with the blaCTX-M-55 gene, and the blaNDM-1, blaTEM, qepA1, and rmtB genes were additionally detected in SECR18-0888. As an extensively drug-resistant E. coli strain producing MCR-1 and NDM-1 was identified in Korea for the first time, continued monitoring of colistin resistance in carbapenem-resistant Enterobacteriaceae should be reinforced.

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray.

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel-coated glass slide and used for profiling of proteins in sera of LC patients in a two-color fluorescence assay. Forty-eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down-regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET).

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

Influence of Mineral Supplementation on the Results from Analysis of Flavonol Glycoside Content in Ginkgo biloba Dietary Supplements.

Flavonoids are a major component of Ginkgo biloba extract (GBE). Several studies have investigated chelate formation and the redox reaction between flavonoids and metal ions; however, the effect of mineral supplements on the results from the analysis of the flavonol glycoside content in products containing GBE dietary supplement remains unknown. In this study, the effects of commonly used mineral supplements on the recovery of quercetin from GBE-containing dietary supplements were investigated using conventional methods of flavonol glycoside determination. Mineral supplements containing Zn (II), Mn (II), and Fe (II) did not affect quercetin recovery, whereas Cu (II) and Fe (III) significantly reduced recovery (P<0.05). Quercetin oxidation was prevented by adding an antioxidant to the diluent (extraction solvent). Among the tested synthetic antioxidants, tert-butyl hydroquinone (TBHQ) promoted the greatest increase in quercetin recovery. The flavonol glycoside content of commercially available GBE-containing dietary supplements was analyzed using a conventional diluent or a diluent containing 20 mg/mL TBHQ. The amount of quercetin recovered from products containing Cu (II) was found to decrease with increasing hydrolysis duration and the duration in the final test solution state using the conventional diluent, while the TBHQ-containing diluent yielded consistent quercetin contents (P<0.05). These findings suggest that quercetin, a major aglycone of GBE flavonol glycosides, can be oxidized by Cu (II) and Fe (III) during the analytical process and, therefore, the total flavonol glycoside content may be underestimated. The addition of TBHQ to the diluent can improve the accuracy and reproducibility of flavonol glycoside content analysis in GBE-containing dietary products supplemented with minerals.

Energy transfer-based multiplexed assay of proteases by using gold nanoparticle and quantum dot conjugates on a surface.

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.

Protein kinase assay on peptide-conjugated gold nanoparticles.

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.

Concentrations of Bisphenols in Canned Foods and Their Risk Assessment in Korea.

The purpose of this study was to survey concentrations of bisphenols in canned foods using liquid chromatography-tandem mass spectrometry, to estimate the dietary exposure to bisphenols, and to assess the related risk for the Korean population from the intake of canned foods. The linearity of bisphenols in the range of 2.5 to 725 µg/L was satisfactory with correlation coefficients ( r2) of 0.999. The limit of detection was 0.14 to 5.85 µg/L, and the limit of quantitation was 0.44 to 17.73 µg/L. Sample recoveries were 70.56 to 113.6%, with relative standard deviations below 10% for spiking levels of 50 and 250 µg/kg (15 and 75 µg/kg for BPS). The bisphenol concentrations in 104 canned foods ranged from undetectable to 1,525 µg/kg. The estimated mean daily intake of bisphenols was 0.54 to 78.69 ng/kg of body weight per day, and the 95th percentile daily intake was 1.92 to 134 ng/kg of body weight per day. Therefore, the intake of bisphenols from canned foods for the population in Korea is unlikely to cause human health problems. The analytical methods used are suitable for regular monitoring and assessment of human exposure to bisphenols from foods.

Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein.

A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation of Ran GTPase-activating protein 1 (RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of sumoylation, fluorescent detection of sumoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner.

Multivariate analysis to discriminate the origin of sesame seeds by multi-element analysis inductively coupled plasma-mass spectrometry.

In this study, inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the concentration of 15 elements (Mg, Al, K, Ca, Cr, Mn, Co, Ni, Cu, Zn, Rb, Sr, Cd, Ba, and Pb) of sesame seeds. Multivariate analysis was then performed to discriminate the origin of sesame seeds. Korean (48), Chinese (44), and Indian (21) samples were used to develop the calibration model. Another 10 samples were used to validate this model. All elements were significantly different (p<0.05) among the samples from three countries, and all elements were subjected to both principal component analysis (PCA) and discriminant analysis. The concentrations of multi-element showed a trend of clustering according to the origin of samples based on PCA. They showed a discrimination rate of 92.0% in the discriminant analysis. The results demonstrated that a combination of ICP-MS multi-element determination and multivariate analysis could be used to discriminate the sesame seed origin.

A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources.

PURPOSE: To evaluate the abilities of these subtyping methods, we distinguished Salmonella Enteritidis (S. Enteritidis) isolated from food products and human clinical samples between 2009 and 2010 in Seoul using five subtyping methods. METHODS: We determined the subtypes of 20 S. Enteritidis isolates from food and human sources using phage typing, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multi-locus sequence typing (MLST). RESULTS: A total of 20 tested isolates were differentiated into six antimicrobial susceptibility patterns, three different phage types, four different PFGE profiles, seven rep-PCR patterns, and one MLST type. Food isolates were considerably more susceptible to antibiotics than human isolates. We were best able to discriminate among S. Enteritidis isolates using rep-PCR, and obtained the highest Simpson's diversity index of 0.82, whereas other methods produced indices that were less than 0.71. PFGE pattern appeared to be more related to antimicrobial resistance and phage types of S. Enteritidis isolates than rep-PCR. MLST revealed identical alleles in all isolates at all seven loci examined, indicating no resolution. CONCLUSION: The results of this study suggest that rep-PCR provided the best discriminatory power for phenotypically similar S. Enteritidis isolates of food and human origins, whereas the discriminatory ability of MLST may be problematic because of the high sequence conservation of the targeted genes.