Development of a Specific and Rapid Diagnostic Method for Detecting Influenza A (H1N1) pdm09 Virus Infection Using Immunochromatographic Assay

AbstractObjectivesThe aim of this study was to develop an immunochromatographic assay (ICA) for the detection of influenza A (H1N1) pdm09 virus infection.Materials and methodsSeveral monoclonal antibodies against influenza A (H1N1) pdm09 virus were generated and an ICA (pdm09-ICA) was developed for the rapid and specific detection of influenza A (H1N1) pdm09 virus infection. The specificity and sensitivity of the developed assay were compared with that of hemagglutination assay and real-time reverse-transcription polymerase chain reaction (rRT-PCR).ResultsThe detection limit was estimated to be 1/2 (8) hemagglutinating unit; the sensitivity and specificity rates of pdm09-ICA were 75.86% (110/145) and 100% (43/43), respectively, compared with rRT-PCR. The cross-reactivity for 20 influenza viruses, including seasonal H1N1 viruses, was found to be negative except for the H1N1 virus (A/Swine/Korea/GC0503/2005).ConclusionThese results indicate that the proposed method can be easily used for rapid and specific detection of the pdm09 infection. The assay developed in this study would be a useful tool for distinguishing the pdm09 infection from seasonal influenza A and B infections

Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways

BACKGROUND: Nitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells. METHODS: Nitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1. RESULTS: The transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (±)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)α and PKCδ from the cytosol to the membrane. Furthermore, the PKC-α,βinhibitors, GÖ6976 (10 nM) and PKCδ inhibitors, rottlerin (4 μM) inhibited the NOR-1 induced migration of PKCα and PKCδ respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCα and PKCδ with ERK1/2 pathways. CONCLUSION: Exogenous NO induced the MUC5AC mucin gene and protein through the PKCα and PKCδ – ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways

Simultaneous electrochemical detection of both PSMA (+) and PSMA (-) prostate cancer cells using an RNA/peptide dual-aptamer probe

Using an RNA/peptide dual-aptamer probe, both PSMA (+) and PSMA (-) prostate cancer cells were simultaneously detected by electrochemical impedance spectroscopy. This approach can be applied as a general tool for early diagnosis of prostate cancer.CATALONA WJ, 1993, JAMA-J AM MED ASSOC, V270, P948Lupold SE, 2002, CANCER RES, V62, P4029Kue PF, 2002, INT J CANCER, V102, P572, DOI 10.1002/ijc.10734Drummond TG, 2003, NAT BIOTECHNOL, V21, P1192, DOI 10.1038/nbt873DARAIN F, 2004, BIOSENS BIOELECTRON, V20, P856Ban CG, 2004, NUCLEIC ACIDS RES, V32, DOI 10.1093/nar/gnh109Ghosh A, 2004, J CELL BIOCHEM, V91, P528, DOI 10.1002/jcb.10661LEVIN MA, 2005, J UROLOGY, V159, P475Rodriguez MC, 2005, CHEM COMMUN, P4267, DOI 10.1039/b506571bZitzmann S, 2005, CLIN CANCER RES, V11, P139Horninger W, 2001, CANCER-AM CANCER SOC, V91, P1667Lang SH, 2001, BRIT J CANCER, V85, P590Yamamoto T, 2001, UROLOGY, V58, P994Palecek E, 2002, CRIT REV ANAL CHEM, V32, P261Narain V, 2002, CANCER METAST REV, V21, P17Edwards S, 2005, BRIT J CANCER, V92, P376, DOI 10.1038/sj.bjc.6602261Postma R, 2005, EUR J CANCER, V41, P825, DOI 10.1016/j.ejca.2004.12.029Cahova-Kucharikova K, 2005, ANAL CHEM, V77, P2920Rahman MA, 2005, ANAL CHEM, V77, P4854, DOI 10.1021/ac050558vCho M, 2006, NUCLEIC ACIDS RES, V34, DOI 10.1093/nar/gkl364Farokhzad OC, 2006, P NATL ACAD SCI USA, V103, P6315, DOI 10.1073/pnas.0601755103Chu TC, 2006, CANCER RES, V66, P5989, DOI 10.1158/0008-5472.CAN-05-4583McNamara JO, 2006, NAT BIOTECHNOL, V24, P1005, DOI 10.1038/nbt1223Palecek E, 1998, BIOSENS BIOELECTRON, V13, P621Min K, 2008, BIOSENS BIOELECTRON, V23, P1819, DOI 10.1016/j.bios.2008.02.021CHO M, 2008, BMB REPORTS, V41, P119Kim D, 2007, J AM CHEM SOC, V129, P7661, DOI 10.1021/ja071471pMaalouf R, 2007, ANAL CHEM, V79, P4879, DOI 10.1021/ac070085nKRAHN MD, 1994, JAMA-J AM MED ASSOC, V272, P773

Scutellaria baicalensis

Antimycin A (AMA) damages mitochondria by inhibiting mitochondrial electron transport and can produce reactive oxygen species (ROS). ROS formation, aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. The present study sought to investigate extracts of Scutellaria baicalensis and its flavonoids (baicalin, baicalein, and wogonin), whether they could protect mitochondria against oxidative damage. The viability of L6 cells treated with AMA increased in the presence of flavonoids and extracts of S. baicalensis. ATP production decreased in the AMA treated group, but increased by 50% in cells treated with flavonoids (except wogonin) and extracts of S. baicalensis compared to AMA-treated group. AMA treatment caused a significant reduction (depolarized) in mitochondrial membrane potential (MMP), whereas flavonoid treatment induced a significant increase in MMP. Mitochondrial superoxide levels increased in AMA treated cells, whereas its levels decreased when cells were treated with flavonoids or extracts of S. baicalensis. L6 cells treated with flavonoids and extracts of S. baicalensis increased their levels of protein expression compared with AMA-treated cells, especially water extracts performed the highest levels of protein expression. These results suggest that the S. baicalensis extracts and flavonoids protect against AMA-induced mitochondrial dysfunction by increasing ATP production, upregulating MMP, and enhancing mitochondrial function

A Case of Neck Abscess Caused by Salmonella Serotype D in a Patient with Liver Cirrhosis

Non-typhoidal salmonellosis, which is increasing nowadays in Korea as well as in the developed countries, is manifested as enteritis in most cases, but it also encompasses bacteremia, intraabdominal infections, and bone, joint and soft tissue infections. These rare diseases are known to result from primary gastrointestinal infection and subsequent bacteremia with or without symptoms. We experienced a case of neck abscess caused by Salmonella serotype D, which is a rare but important differential diagnosis of neck abscess. We herein report it

In Vivo Biocompatibility Study of Electrospun Chitosan Microfiber for Tissue Engineering

In this work, we examined the biocompatibility of electrospun chitosan microfibers as a scaffold. The chitosan microfibers showed a three-dimensional pore structure by SEM. The chitosan microfibers supported attachment and viability of rat muscle-derived stem cells (rMDSCs). Subcutaneous implantation of the chitosan microfibers demonstrated that implantation of rMDSCs containing chitosan microfibers induced lower host tissue responses with decreased macrophage accumulation than did the chitosan microfibers alone, probably due to the immunosuppression of the transplanted rMDSCs. Our results collectively show that chitosan microfibers could serve as a biocompatible in vivo scaffold for rMDSCs in rats

A single gene of a commensal microbe affects host susceptibility to enteric infection

Indigenous microbes inside the host intestine maintain a complex self-regulating community. The mechanisms by which gut microbes interact with intestinal pathogens remain largely unknown. Here we identify a commensal Escherichia coli strain whose expansion predisposes mice to infection by Vibrio cholerae, a human pathogen. We refer to this strain as 'atypical' E. coli (atEc) because of its inability to ferment lactose. The atEc strain is resistant to reactive oxygen species (ROS) and proliferates extensively in antibiotic-treated adult mice. V. cholerae infection is more severe in neonatal mice transplanted with atEc compared with those transplanted with a typical E. coli strain. Intestinal ROS levels are decreased in atEc-transplanted mice, favouring proliferation of ROS-sensitive V. cholerae. An atEc mutant defective in ROS degradation fails to facilitate V. cholerae infection when transplanted, suggesting that host infection susceptibility can be regulated by a single gene product of one particular commensal species.

Direct Reprogramming of Rat Neural Precursor Cells and Fibroblasts into Pluripotent Stem Cells

Background: Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes. Methodology and Principal Findings: iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency. Conclusions/Significance: Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease

Identification of a Novel Single Nucleotide Polymorphism of HADHA Gene at a Referred Primer-binding Site During Pre-diagnostic Tests for Preimplantation Genetic Diagnosis

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene disorder