Clinical evaluation of noninvasive prenatal testing for sex chromosome aneuploidies in 9,176 Korean pregnant women: a single-center retrospective study.

BACKGROUND: To evaluate the clinical significance of noninvasive prenatal testing (NIPT) for detecting fetal sex chromosome aneuploidies (SCAs) in Korean pregnant women. METHODS: We retrospectively analyzed NIPT data from 9,176 women with singleton pregnancies referred to the CHA Biotech genome diagnostics center. Cell-free fetal DNA (cffDNA) was extracted from maternal peripheral blood, and high-throughput massively parallel sequencing was conducted. Subsequently, the positive NIPT results for SCA were validated via karyotype and chromosomal microarray analyses. RESULTS: Overall, 46 cases were SCA positive after NIPT, including 20, 12, 8, and 6 for Turner, triple X, Klinefelter, and Jacob syndromes, respectively. Among 37 women with invasive prenatal diagnosis, 19 had true positive NIPT results. The overall positive predictive value (PPV) of NIPT for detecting SCAs was 51.35%. The PPV was 18.75% for Turner, 88.89% for triple X, 71.43% for Klinefelter, and 60.00% for Jacob's syndromes. NIPT accuracy for detecting sex chromosome trisomies was higher than that for sex chromosome monosomy (P = 0.002). No significant correlation was observed between fetal SCA incidence and maternal age (P = 0.914), except for the borderline significance of Jacob's syndrome (P = 0.048). No significant differences were observed when comparing NIPT and karyotyping validation for fetal SCA according to pregnancy characteristics. CONCLUSION: Our data suggest that NIPT can reliably screen for SCAs, and it performed better in predicting sex chromosome trisomies compared with monosomy X. No correlation was observed between maternal age and fetal SCA incidence, and no association was observed between different pregnancy characteristics. The accuracy of these findings requires improvements; however, our study provides an important reference for clinical genetic counseling and further management. Larger scale studies, considering confounding factors, are required for accurate evaluation.

Distinctive HBV Replication Capacity and Susceptibility to Tenofovir Induced by a Polymerase Point Mutation in Hepatoma Cell Lines and Primary Human Hepatocytes.

Tenofovir disoproxil fumarate (TDF) has been regarded as the most potent drug for treating patients with chronic hepatitis B (CHB). However recently, viral mutations associated with tenofovir have been reported. Here, we found a CHB patient with suboptimal response after more than 4 years of TDF treatment. Clonal analysis of hepatitis B virus (HBV) isolated from sequential sera of this patient identified the seven previously reported TDF-resistant mutations (CYELMVI). Interestingly, a threonine to alanine mutation at the 301 amino acid position of the reverse-transcriptase (RT) domain, (rtT301A), was commonly accompanied with CYELMVI at a high rate (72.7%). Since the rtT301A mutation has not been reported yet, we investigated the role of this naturally occurring mutation on the viral replication and susceptibility to tenofovir in various liver cells (hepatoma cells as well as primary human hepatocytes). A cell-based phenotypic assay revealed that the rtT301A mutation dramatically impaired the replication ability with meaningful reduction in sensitivity to tenofovir in hepatoma cell lines. However, attenuated viral replication by the rtT301A mutation was significantly restored in primary human hepatocytes (PHHs). Our findings suggest that the replication capability and drug sensitivity of HBV is different between hepatoma cell lines and PHHs. Therefore, our study emphasizes that validation studies should be performed not only in the liver cancer cell lines but also in the PHHs to understand the exact viral fitness under antiviral pressure in patients.

Expansion of Shiga Toxin-Producing Escherichia coli by Use of Bovine Antibiotic Growth Promoters.

Antibiotics are routinely used in food-producing animals to promote growth and prevent infectious diseases. We investigated the effects of bovine antibiotic growth promoters (bAGPs) on the propagation and spread of Shiga toxin (Stx)-encoding phages in Escherichia coli. Co-culture of E. coli O157:H7 and other E. coli isolated from cattle in the presence of sublethal concentrations of bAGPs significantly increased the emergence of non-O157, Stx-producing E. coli by triggering the SOS response system in E. coli O157:H7. The most substantial mediation of Stx phage transmission was induced by oxytetracyline and chlortetracycline, which are commonly used in agriculture. bAGPs may therefore contribute to the expansion of pathogenic Stx-producing E. coli.

Novel adjuvant strategy to potentiate bacitracin against MDR MRSA.

OBJECTIVES: Bacitracin is an antimicrobial peptide that is frequently used as an active ingredient in antimicrobial ointments. However, bacitracin resistance is highly prevalent in community-associated MRSA (CA-MRSA) strains and significantly compromises the effectiveness of existing antimicrobial ointments. In this study, we aimed to develop novel adjuvants to enhance the antimicrobial activity of bacitracin by using alkyl gallates. METHODS: The growth of MRSA USA300, the predominant CA-MRSA strain in the USA, was determined in the presence of bacitracin and alkyl gallates at various concentrations. The viability of USA300 and MDR clinical isolates of MRSA was measured after exposure to various combinations of bacitracin and alkyl gallates. RESULTS: Whereas 100 U/mL bacitracin did not inhibit USA300, 1 U/mL bacitracin in combination with as low as 2 mg/L octyl gallate (OG) and 8 mg/L dodecyl gallate (DG), respectively, completely inhibited the growth of USA300. Among the tested alkyl gallates, OG most significantly enhanced the bactericidal activity of bacitracin. For example, 10(-3) U/mL bacitracin with 5 mg/L OG effectively killed USA300, which is an ∼200 000-fold decrease in the MBC of bacitracin for USA300. Furthermore, bacitracin/OG combinations demonstrated similar levels of antimicrobial activity against human clinical isolates of MRSA resistant to multiple antibiotics of clinical importance. CONCLUSIONS: Some alkyl gallates, particularly OG, significantly increased the antimicrobial activity of bacitracin against MDR MRSA.

Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.

Brain inositol is a novel stimulator for promoting Cryptococcus penetration of the blood-brain barrier.

Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.

Cryptococcus neoformans activates RhoGTPase proteins followed by protein kinase C, focal adhesion kinase, and ezrin to promote traversal across the blood-brain barrier.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.

Cell-Free Fetal DNA Screening Analysis in Korean Pregnant Women: Six Years of Experience and a Retrospective Study of 9327 Patients Analyzed from 2017 to 2022.

Cell-free DNA (cfDNA) screening for normal fetal aneuploidy has been widely adopted worldwide due to its convenience, non-invasiveness, and high positive predictive rate. We retrospectively evaluated 9327 Korean women with single pregnancies who underwent a non-invasive prenatal test (NIPT) to investigate how various factors such as maternal weight, age, and the method of conception affect the fetal fraction (FF). The average FF was 9.15 ± 3.31%, which decreased significantly as the maternal body mass index (BMI) increased (p < 0.001). The highly obese group showed a 'no-call' rate of 8.01%, which is higher than that of the normal weight group (0.33%). The FF was 8.74 ± 3.20% when mothers were in their 40s, and lower than that when in their 30s (9.23 ± 3.34, p < 0.001) and in the natural pregnancy group (9.31% ± 3.33). The FF of male fetuses was observed to be approximately 2.76% higher on average than that of female fetuses. As the gestational age increased, there was no significant increase in the fraction of fetuses up to 21 weeks compared to that at 10-12 weeks, and a significant increase was observed in the case of 21 weeks or more. The FFs in the NIPT high-risk result group compared to that in the low-risk group were not significantly different (p = 0.62). In conclusion, BMI was the factor most associated with the fetal fraction. Although the NIPT is a highly prevalent method in prenatal analysis, factors affecting the fetal fraction should be thoroughly analyzed to obtain more accurate results.

Repression of flagella motility in enterohemorrhagic Escherichia coli O157:H7 by mucin components.

Whole genome-scale transcriptome analysis of enterohemorrhagic Escherichia coli (EHEC) O157:H7 EDL933 was performed to investigate the influence of mucin components on the EHEC gene expression. Here we report that the 732 candidate genes were differentially expressed by the presence of 0.5% porcine stomach mucin, including the 8 flagella-related genes. Quantitative real-time PCR analyses revealed that the transcription expression of the flg genes (encoding the structural components for flagella basal body) was down-regulated by the mucin components. Indeed, bacterial swarming motility was drastically reduced when grown on 0.3% trypton agar plates containing the mucin. These results imply that gastrointestinal (GI) mucin is a possible environmental signal which negatively regulates the flagellation of EHEC O157:H7 in the GI tract.

The effect of the surface coating of human adipose-derived stem cells by various GAGs on the biodistribution of them upon intravenous administration.

Glycosaminoglycans (GAGs) are essential for cell-cell and cell-ECM interactions. Unique structures of GAGs provide high affinities to specific cell receptors. Especially, hyaluronic acid (HA), chondroitin sulfate (CS), and heparin are known to have affinities to the liver sinusoidal endothelial cells (LSECs), so they have been utilized as a ligand for liver targeting nanoparticle systems. In this study, we compared different GAGs as a targeted cell delivery ligand by using lipid-conjugated GAGs. Conjugated lipids of GAGs could provide a stable coating over 2 days on the surface of human adipose-derived stem cells (hADSCs) by physical insertion. The hADSCs coated by different GAGs were intravenously injected into mice, and the biodistribution of cells was analyzed by an In Vivo Imaging System (IVIS) to compare the effect of various GAGs on the modulation of biodistribution of stem cells. The results showed that all three GAGs could provide less entrapment in the lung but enhanced accumulation in the liver and spleen. Especially, HA- and heparin coating on hADSCs showed a 1.5-fold higher accumulation than CS-coating on hADSCs in the liver and spleen. Thus, lipid-conjugated HA and heparin are potentially useful coating materials for the liver or spleen-targeted delivery system of therapeutic stem cells.

Susceptibility of Drug Resistant Hepatitis B Virus Mutants to Besifovir.

Currently, interferon alpha and nucleos(t)ide analogues (NAs) are clinically available to treat hepatitis B virus (HBV) infection. Several NAs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV) and tenofovir (TDF or TAF) have been approved and administered to chronic hepatitis B (CHB) patients. NAs inhibit HBV DNA synthesis by targeting the reverse transcriptase (RT) domain of HBV polymerase. Several mutations in the RT domain which lead to drug resistance against NAs have been reported, even for TDF and TAF which are highly potent with very low resistance rate. Besifovir (BFV) is a new antiviral dGMP analogue able to be used as a new NA drug for the control of CHB infection. Drug resistance to BFV is not well known due to its shorter duration of clinical use. Recently, we reported that rtL180M (M) and rtM204V (V) mutations, already resistant to LMV, are associated with BFV resistance. However, the susceptibility to BFV of previously known HBV mutants resistant to various drugs has not been studied. To investigate this, we performed in vitro drug susceptibility assays using natural and artificial mutants that are associated with resistance to LMV, ADV, ETV or TDF. As a result, LMV-resistant mutants were not susceptible to BFV and ETV-resistant clones showed partial resistance against BFV as well. However, ADV-resistant mutants were highly sensitive to BFV. In case of tenofovir-resistant mutations, the HBV mutants harboring primary mutations to tenofovir resistance were susceptible to BFV. Therefore, our study revealed that BSV may serve as an alternative drug for patients with ADV-, ETV-, TDF- or TAF-resistance.

Identification and Characterization of Besifovir-Resistant Hepatitis B Virus Isolated from a Chronic Hepatitis B Patient.

Hepatitis B virus (HBV) is known to cause severe liver diseases such as acute or chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B (CHB) infection is a major health problem with nearly 300 million individuals infected worldwide. Currently, nucleos(t)ide analogs (NAs) and interferon alpha are clinically approved treatments for HBV infection. NAs are potent antiviral agents that bind to HBV polymerase and block viral reverse transcription and replication. Besifovir dipivoxil maleate (BSV) is a newly developed NA against HBV in the form of acyclic nucleotide phosphonate that is available for oral administration similar to adefovir and tenofovir. Until now, resistance to BSV treatment has not been reported. In this study, we found a CHB patient who showed viral breakthrough after long-term treatment with BSV. The isolated HBV DNA from patient's serum were cloned into the replication-competent HBV 1.2 mer and the sequence of reverse transcriptase (RT) domain of HBV polymerase were analyzed. We also examined the drug susceptibility of generated clones in vitro. Several mutations were identified in HBV RT domain. A particular mutant harboring ten RT mutations showed resistance to BSV treatment in vitro. The ten mutations include rtV23I (I), rtH55R (R), rtY124H (H), rtD134E (E), rtN139K (K), rtL180M (M), rtM204V (V), rtQ267L (L), rtL269I (I) and rtL336M (M). To further identify the responsible mutations for BSV resistance, we performed in vitro drug susceptibility assay on several artificial clones. As a result, our study revealed that rtL180M (M) and rtM204V (V) mutations, already known as lamivudine-resistant mutations, confer resistance to BSV in the CHB patient.

Hepatic control elements promote long-term expression of human coagulation factor IX gene in hydrodynamically transfected mice.

BACKGROUND: Long-term expression of the delivered target gene is critical for successful gene therapy. Recently, hepatic control region I (HCR I) originating from the apolipoprotein (apo)C-I pseudogene was shown to be a critical element for long-term gene expression in the liver of mice. HCR II is another hepatic control region of apoC-I. METHODS: HCR I, HCR II and HCR I/II-containing plasmids encoding factor IX were prepared and hydrodynamically transferred into the liver of normal and hemophilia B mice. Factor IX expression, clotting activity and formation of antibodies against the expressed gene product were compared. RESULTS: HCR I-, HCR II- and HCR I/II-containing plasmids all induced long-term gene expression in both normal and hemophilia B mice. Post-transfection factor IX expression in the hemophilia B mice remained above 500 ng/ml for 210 days. Antibodies against human factor IX were detected at a low level in the serum, although they had no effect on the levels and clotting activity of the expressed factor IX. CONCLUSIONS: We have shown in mouse models that hydrodynamic transfection of pBS-HCRII-HP-FIXA and pBS-HCRI/II-HP-FIXA was able to induce and maintain the expression and clotting activity of human factor IX for a long period of time at a potentially therapeutic level. With an appropriate delivery system, this type of plasmid vector could be clinically useful for the hepatic expression of therapeutic genes including human factor IX.

Nanoparticle formulation for controlled release of capsaicin.

Capsaicin might be an effective pharmacological agent for the treatment of discogenic back pain due to its effect on pain control neuronal degeneration. Therefore, capsaicin-loaded nano- and micro-particles for sustained release were formulated by nano-precipitation or oil-in-water single emulsion solvent evaporation/extraction method. First, the capsaicin-loaded PLGA nanoparticles were prepared by nano-precipitation method. By increasing the volume of oil-water ratio from 1:2 to 1:5, slight changes in size from 162 +/- 3 nm to 153 +/- 3 nm and in drug loading efficiency from 25% to 20% were observed, whereas the drug release period was significantly changed from 11 days for 1:2 to 5 days for 1:5 ratio. To get a more sustained release, a modified single emulsion method was applied with three kinds of biocompatible polymers (PLLA, PLGA, and PCL). Among them, PLLA particles showed a much sustained release profile than PLGA or PCL ones with the similar size. For PLLA particles, particles size and drug encapsulation efficiency increased as the oil/water ratio decreased, and the bigger particles showed the slower release profiles as well as the higher drug-loading efficiency, thus about 1 month release was obtained with 800 nm particles. In conclusion, formulation for the controlled release of capsaicin from 1 week to 1 month was prepared by using biocompatible nanoparticles.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

Characterization of BNT2, an intrinsically curved DNA of Escherichia coli O157:H7.

The gene regulation by intrinsically curved DNA is one way for bacterial sensing of and response to environmental changes. Previously, we showed that the genetic element BNT2 upstream of the ecf (eae-positive conserved fragment) operon in the Escherichia coli O157:H7 virulence plasmid (pO157) has characteristics typical of intrinsically curved DNA, including the presence of multi-homopolymeric adenine:thymine tracts (AT tracts) and electrophoretic anomaly at 4 degrees C. Here we report that a local intrinsic curvature induced by the two phased AT tracts within the unusual promoter sequence of BNT2 played a major role for its temperature-dependent promoter activity. The base substitution of the AT tract in the spacer DNA between the -35 and the unusual -10 regions of the BNT2 promoter with non-AT tract sequence reduced intrinsic curvature slightly at 4 degrees C, but greatly affected its transcriptional activity. This implies that such a local intrinsic curvature within the unusual promoter of BNT2 is important for thermoregulation of the ecf operon.

Omics-based analysis of the luxS mutation in a clinical isolate of Escherichia coli O157:H7 in Korea.

The purpose of this study was to investigate the relationship between the global regulatory mechanism known as quorum sensing and expression of virulence factors in Escherichia coli O157:H7. A non-polar luxS deletion was introduced into the chromosome of strain CI03J, a human clinical isolate from South Korea, to create the DeltaluxS mutant strain ML03J. Phenotypic characterization of wild-type and mutant strains demonstrated that ML03J had no obvious growth or metabolic defects on 0.2% glucose LB medium, produced a functionally-defective flagellum, and could not utilize sorbose; the biological significance of sorbose utilization is unknown. Omics-based analysis revealed the involvement of LuxS in the transcriptional activation of several flagella/chemotaxis-related genes (flhD; fliA, C, D, S, Z; cheA, Y, and Z), repression of glutamate-dependent acid resistance genes (gadAB), and expression of virulence factors including Shiga toxin, hemolysin, and SepD within the LEE pathogenicity island.

Intramolecular distance in the conjugate of urate oxidase and fatty acid governs FcRn binding and serum half-life in vivo.

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)-mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA-conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins.

Atmospheric pressure chemical ionization and fragmentation of aminomonosaccharides in H2O and D2O.

Aminomonosaccharides (glucosamine, galactosamine, and mannosamine) in H2O and D2O were ionized by atmospheric pressure chemical ionization (APCI) and their fragmentation patterns were investigated to identify them. All the aminomonosaccharides showed the same fragment ions but their relative ion intensities were different. Major product ions generated in H2O were [M + H]+, [M + H - H2O]+, and [2M + H - 3H2O]+, while in D2O were [M(D6) + D]+, [M(D6) + D - D2O]+, and [2M(D6) + D - D2O - 2HDO]+. At a high fragmentor voltage above 120 V, the relative ion intensities of the major product ions showed different trends according to the aminomonosaccharides. For the use of H2O as solvent and eluent, the order of the ion intensity ratio of [M + H - H2O]+/[2M + H - 3H2O]+ was galactosamine > mannosamine > glucosamine. When using D2O as solvent and eluent, the order of the ion intensity ratios of [M(D6) + D - D2O]+/[MD6 + D]+ and [2M(D6) + D - D2O - 2HDO]+/[M(D6) + D]+ was mannosamine > galactosamine > glucosamine. It was found that glucosamine, galactosamine, and mannosamine could be distinguished by the specific trends of the major product ion ratios in H2O and D2O.

Detection of cytolethal distending toxin and other virulence characteristics of enteropathogenic Escherichia coli isolates from diarrheal patients in Republic of Korea.

Cytolethal distending toxins (CDTs) represent an emerging family of newly described bacterial products that are produced by a number of pathogens. The genes encoding these toxins have been identified as a cluster of three adjacent genes, cdtA, cdtB, and cdtC, plus 5 cdt genetic variants, designated as cdt-I, cdt-II, cdt-III, cdt-IV, and cdt- V, have been identified to date. In this study, a general multiplex PCR system designed to detect Escherichia coli cdts was applied to investigate the presence of cdt genes among isolates. As a result, among 366 E. coli strains, 2.7% were found to carry the cdtB gene. In addition, the use of type-specific primers revealed the presence of cdt-I, cdtIV, and cdt-V types of the cdt gene, yet no cdt-II or cdt- III strains. The presence of other virulence genes (stx1, stx2, eae, bfp, espA, espB, and espD) was also investigated using a PCR assay. Among the 10 cdtB gene-positive strains, 8 were identified as CDT-producing typical enteropathogenic E. coli (EPEC) strains (eae(+), bfp(+)), whereas 2 were identified as CDT-producing atypical EPEC strains (eae(+), bfp(-)). When comparing the cytotoxic activity of the CDT-producing typical and atypical EPEC strains, the CDT-producing atypical EPEC strains appeared to be less toxic than the CDT-producing typical EPEC strains.