Appl. Biol. Chem., Tokyo Univ. of Agri.; 2Dept. Pathol. Inst. Dev. Res. Aichi Human Service Ctr.; 3School of Cultural Creative Studies, Aoyama Gakuin Univ.; 4Nagahama Inst. Bio-Sci. Tech.; 5School of Human Cultures, Univ. of Shiga Pref. Introduction: Quinolinic acid which is

known to be neurotoxic and uremic is an intermediary metabolite in kynurenine pathway. Erythropoietin (EPO) is a Navitoclax concentration hormone produced by the kidney that leads to the formation of red blood cell. Renal anemia has recognized as one of complications of chronic kidney disease, which is mediated by the reduced production of erythropoietin derived by renal fibrosis. It has been PD-0332991 order reported the influence of Quinolinic acid and 3-hydroxykynurenine, the metabolites in kynurenine pathway, on EPO synthesis, but its details are enigma.1)2)

The aim of this study is to investigate the effect of Quinolinic Acid on renal fibrosis and erythropoietin expression using QPRT knockout mice which are able to artificially accumulate Quinolinic Acid. Methods: DNA Microarray was used to evaluate gene expression in the kidney of wild type and QPRT knockout mice. The collagen deposition was determined by Sirius red staining. The mRNA expression of EPO, collagen-type-1-alpha-1 (col1a1), and Hif2a were measured by real-time PCR. And the levels of hemoglobin and hematocrit were measured. Results: The microarray data indicate that gene families involved in fibrosis and transporter were upregulated in QPRT Knockout. In QPRT knockout Cyclin-dependent kinase 3 mice, Col1a1 mRNA level and collagen deposition were increased, suggested QPRT depletion have an effect on renal fibrosis. And, QPRT knockout mice significantly decreased

EPO mRNA expression (p < 0.05), hemoglobin (p < 0.01), and hematocrit (p < 0.05). Conclusion: Our results suggested that quinolinic acid accumulation in the kidney initiates renal fibrosis, and decreases EPO synthesis. 1) Pawlak D, Koda M, Pawlak S, Wolczynski S, Buczko W., Contribution of quinolinic acid in the development of anemia in renal insufficiency. Am J Physiol Renal Physiol. 284(4):F693–700. (2003) 2) Pawlak D, Koda M, Wolczynski S, Buczko W., Mechanism of inhibitory effect of 3-hydroxykynurenine on erythropoiesis in patients with renal insufficiency. Adv Exp Med Biol., 527:375–380 (2003).

These activated B-1 B cells are then able to produce antigen-specific IgM antibodies in vivo [10]. We note that stimulatory lipids may not be entirely unique to post-sensitization livers, as the response induced by iNKT cell stimulation with lipids from livers of naïve mice was greater than the baseline response (Groups E versus B, Fig. 1A,B). Thus, there may be a background level of iNKT cell stimulation by endogenous lipids, which is consistent with prior descriptions of partially activated iNKT cells

in naïve mice. Alternatively, this observation may represent iNKT cell activation from background exposure to microbial components such as cell wall glycosylceramides. Potential activation by the murine microbiota would not detract from the results, however, as the same degree of enhancement would be seen GSK126 in all

experimental groups. Adoptive transfer of LMNC from wild-type mice can reconstitute CS in CD1d-deficient mice. We show that CD1d itself is essential, based on experiments involving anti-CD1d-blocking antibody. However, background BGJ398 in vivo expression of CD1d in recipient mice is not necessary for CS reconstitution. We conclude that the transferred iNKT cells are sufficiently activated in vitro. By extension, LMNC are inferred to be presenting lipid antigen via CD1d, thereby activating iNKT cells. Candidate APC include hepatic dendritic cells [31] and iNKT cells themselves [32]. Although hepatocytes seem well suited to serve as essential APC for the presentation of lipid antigens to iNKT cells, our results suggest that they are not essential. APC amongst LMNC are sufficient. Following adoptive transfer, activated donor iNKT cells do not home to the recipient liver at 1 day yet are able to reconstitute CS. We performed

this experiment in Jα18−/− and CD1d−/− mice, both strains of which are iNKT cell deficient, with the same result. Hepatocytes in Jα18−/− mice express CD1d, but this potential to present glycolipids to iNKT cells did not appear to lure donor iNKT cells. Reconstituted CS therefore appears to represent a slightly different phenomenon than wild-type CS, despite phenotypic similarities. We conclude that extrahepatic activation of iNKT cells occurs in reconstituted mice, an important consideration Adenosine in the future utilization of this mouse model for understanding iNKT cell biology. Despite these revelatory data, we still contend that the liver is an essential site for iNKT cell activity, based on our prior work. We have previously shown that actively sensitized mice double their percentage of hepatic iNKT cells (as measured by tetramer binding) within 2 h after sensitization, likely a reflection of both an increase in numbers and activation [9]. We have shown in wild-type mice that very early after sensitization, hepatic iNKT cells express IL-4 and not IFN-γ [10].

1). Splenic lymphocytes from mice

immunized with AMH formulated Dasatinib solubility dmso with adjuvants DDA and BCG PSN secreted high levels of IFN-γ upon stimulation with Ag85B, HspX, Mpt64190–198 and PPD (Fig. 2). Splenic lymphocytes from mice immunized with AMH produced higher level of IFN-γ than those immunized with Ag85B, AMM, BCG and PBS with the stimulation of HspX, Mpt64190–198 and PPD. When stimulated with antigen Ag85B, the level of IFN-γ induced by AMH vaccine was lower than that by AMM (P < 0.05) and Ag85B (P > 0.05) vaccines, but was still higher than that receiving BCG (P < 0.05). With the aid of adjuvant DDA + BCG PSN, AMH induced higher levels of antigen-specific IgG1 and IgG2a than Ag85B and AMM (Table 1). Ag85B-specific IgG2a and HspX-specific IgG1 and IgG2a from AMH group were the highest among all groups. PPD-specific IgG1 and IgG2a from the mice immunized with AMH were higher than Ag85B and BCG group. The ratio of Ag85B-specific IgG2a/IgG1 from AMH group was lower than that of BCG group but higher than that of AMM and Ag85B groups. The ratio of HspX-specific IgG2a/IgG1 from AMH group was the highest among all groups. High IgG2a/IgG1 ratio reflects Th1 activity which produces IFN-γ to promote intracellular killing activity by activating

macrophages and cytotoxic T cells [17]. Cell-mediated immune responses in mice primed with BCG and boosted by AMH, AMM, or AMM + AMH were analysed with the stimulation of Ag85B and PPD. The results showed that there were higher levels of IFN-γ Casein kinase 1 production selleck chemicals llc in mice boosted with AMH, AMM and AMM + AMH vaccines than the group of BCG (Fig. 3). It

indicated that Ag85B-, PPD-specific cell-mediated immunity were highly induced by AMH, AMM and AMM + AMH boosting. Unlike the fusion proteins, single-protein Ag85B boosting did not significantly induce high cell-mediated immunity compared with BCG alone. There was no significant difference among AMM, AMH and AMM + AMH groups. The boost with subunit vaccines induced a higher humoral immune response against Ag85B (data not shown). PBS control did not produce antibodies. The titres of IgG1 and IgG2a against Ag85B from mice immunized with BCG and boosted with subunit vaccines were higher than that primed with BCG alone (P < 0.05), whereas there were no significant differences among boosting groups. Protective efficacy was evaluated by CFU count in mice boosted with different protein vaccines followed by challenging with virulent M. tuberculosis H37Rv. The CFUs from the lungs of mice boosted with the subunit vaccines AMM + AMH and AMM were significantly lower than PBS injection, although AMH subunit vaccine boosting did not lead to a significant decrease in CFUs. The bacilli were effectively inhibited in the lungs of mice boosted by AMM + AMH in DDA-BCG PSN, which even induced significantly lower CFU than BCG group (P < 0.05) (Fig. 4).

This divergence probably results from the different infectious disease challenges associated with the respective ecological niches that learn more these two species inhabit. Unfortunately, these differences between the mouse and human immune systems also result in dissimilar inflammatory responses to burns, trauma, and endotoxemia at the gene expression level, such as integrin, ICOS-ICOSL, CD28, and PKCΘ signaling [3]. Therefore, alternatives to classical mouse models, which more closely model human immune system behavior during infection

in vivo, would be of significant benefit for the development of immunomodulatory treatments. The category of new models, which comes closest to achieving this goal, is mice with reconstituted human immune system components. These mice are mainly generated by neonatal injection of human hematopoietic progenitor cells in mice that lack murine innate and adaptive lymphocytes, namely NOD-scid γc−/− (NSG), NOD-scid γctm1sug, NOD Rag1−/− γc−/−, or BALB/c Rag2−/− γc−/− (BRG) mice [4] (Fig. 1). For some studies, a fetal organoid of liver and thymic tissue is implanted under the kidney capsule, which together with the i.v. injection of human hematopoietic progenitor cells generates BM liver thymic mice [5]. In

all of these models, cellular components of the human immune system develop over several months, selleck chemicals llc including human T cells, B cells, natural killer (NK) cells, monocytes, macrophages, and dendritic cells (DCs) [6-8]. However, the degree of human immune system component reconstitution differs significantly between these mouse strains, with 60% of mononuclear cells being of human origin in the spleen and blood of NSG, NOD-scid γctm1sug, and NOD Rag1−/− γc−/− mice 3 months after

human hematopoietic progenitor cell transfer, while in BRG mice only 20% are of human origin at this time point [9, 10]. This difference in the proportion of mononuclear MTMR9 cells of human origin among the various mouse models results at least in part from the polymorphism among mouse strains in signal regulatory protein-α (SIRP-α), an inhibitory receptor on mouse myeloid cells. This receptor recognizes human CD47 in the NOD mouse background and thereby prevents phagocytosis of human cells by the mouse myeloid compartments, which are still intact in all these mouse backgrounds [11]. Indeed, when human or NOD-mouse signal regulatory protein-α is transgenically introduced into BRG mice, or when BRG mice are reconstitute with human hematopoietic progenitor cells that are transduced to express mouse CD47, human immune system reconstitution is similar to that in NSG mice [12, 13]. In particular, human T-cell and NK-cell reconstitution is very sensitive to optimal reconstitution of the other human immune compartments, such as dendritic cells, but comprise up to 60 and 5% of human CD45-positive cells, respectively [9, 14, 15].

Since 2007, GWAS have increasingly been applied to pharmacogenetics to identify loci that affect https://www.selleckchem.com/p38-MAPK.html either drug response or susceptibility to adverse drug reactions. These studies have shown the value of this approach in many fields [18, 78-83]. However, there are limitations in conducting GWAS in pharmacogenetics. First, the variation in drug response is likely to be multifactorial, with many genes working in conjunction with the environment. Second, current GWAS are targeted at elucidating the independent effects of single genes, and may miss interactive or synergistic effects. Furthermore, the challenges in performing adequate replication studies have to be considered for

GWAS in pharmacogenetics, particularly Alisertib when evaluating small cohorts, such as nonresponders to UDCA in PBC. UDCA, which is currently the only available drug in PBC, is thought to work on the downstream events of the pathogenic mechanism of the disease, through reducing the toxicity of bile and reducing bile duct cell apoptosis [84]. There are ongoing studies, focused on exploring, with a GWA approach, the mechanism(s) beyond the lack of biochemical response to UDCA treatment. A major aim of this ongoing project is to identify potential sites for therapeutic intervention in nonresponsive patients.

New therapeutic targets that may be highlighted by GWAS, as applied to pharmacogenetics, can be localized either in the upstream or downstream processes of PBC pathogenesis; from the mechanisms that lead to loss of tolerance to the fibrotic phase secondary to cholestasis. Furthermore, improved knowledge of the genetic basis of the lack of response to UDCA will allow to identify

nonresponders at an early stage and to select them for next-generation drug trials. Attempting to predict the onset and progression of disease is one of the cornerstones of epidemiology. GWAS show significant potential to identify molecular factors that enable patient stratification and might prove useful in personalized medicine. Accurate risk prediction can enable targeted preventative treatments or more intensive follow-up, particularly for patients at high risk of progression. The success of recent GWAS has rapidly changed the outlook Janus kinase (JAK) for genetic risk prediction. These studies have unlocked thousands of clearly validated genetic associations to complex diseases, but their generally weak effects have left their predictive value and clinical utility subject to hot debate. GWAS data might find ready application in risk prediction in PBC in those patients identified at an early stage of the disease. Risk stratification at an early stage may be important from the perspective of developing treatments that either prevent disease entirely or that improve the outcome when instituted before biliary fibrosis and cirrhosis develop.

After a total culture period of 6 h, cells were collected and stained with anti-CD49b Barasertib in vitro and anti-CD3. Cells were permeabilized in Cytofix/Cytoperm reagent and stained with anti-IFN-γ mAb. A standard 4-h 51Cr

release assay was used to assess NK cell cytotoxicity against YAC-1 target cells. YAC-1 cells (ECACC, Salisbury, UK) (106) were labelled with 100 μCi 51Cr (Perkin Elmer, Massachusetts, USA) at 37°C, 5% CO2, for 1.5 h. Freshly isolated hepatic leukocytes or DX5-enriched splenocytes were used as effector cells. For the measurement of cytotoxicity by cytokine-stimulated NK cells, DX5-enriched splenocytes were cultured for 48 h with 1000 U/mL IL-2 (R&D Systems). Hepatic leukocytes were cultured for 48 h with 50 ng/mL IL-15 (R&D Systems) and 2 ng/mL IL-12 (R&D Systems). Cells were plated in a V-bottomed 96-well microtitre plate at 103 target cells per well and various cell numbers of freshly isolated or cytokine-stimulated effector cells. Plates were incubated at 37°C, 5% CO2, for 4 h. Supernatant was TSA HDAC purchase harvested and counted in a 1450 Microbeta Plus Liquid Scintillation Counter (Perkin Elmer) to determine cytotoxicity. Percent specific lysis was calculated as follows:

100×[(experimental release − spontaneous release)/(total release − spontaneous release)]. Staining with anti-NK1.1, anti-CD122 and anti-CD3 was performed to determine the percentage of NK cells in the effector samples. Presented results of specific lysis were recalculated for NK:target cell ratios. All statistical analysis was performed with SPSS 15 software (SPSS, Chicago, IL, USA). The Kolmogorov–Smirnov test indicated that all datasets were not in accordance with a normal distribution (p<0.05). Therefore, the non-parametric Mann–Whitney U test was used. Values of p<0.05 were considered significant. If the assay involved more than two sample populations, multi-variate analysis was performed with the non-parametric Kruskal–Wallis test, in which H values >0.05 either indicated that the samples did not come from identical populations. A Dunnett T3 test was applied to further indicate which sample

populations were significantly different from the others. Values of p<0.05 were considered significant. This work was supported by the Fund for Scientific Research-Flanders and the Foundation against Cancer, a foundation of public interest (G. L.). V.D.C. and T.T. are supported by the Fund for Scientific Research-Flanders, S.T. is supported by the Institute for the Promotion of Innovation by Science and Technology, Flanders, Belgium. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. "
“The virulence of Staphylococcus epidermidis is related to its capacity to form biofilms.

The age-specific prevalence of patients with ESKD was estimated using a logistic regression model with generalized estimating

equation based on the data of high-income countries. The ratio between number GDC-0941 in vivo of RRT and estimated number of ESKD (RRT/ESKD ratio) \ were computed for all countries on the basis of gross national income levels for each country. Results: The number of patients with ESKD was estimated to be 7.8 million, of which 2.3 million (30%) had access to RRT, leading to 5.5 million preventable deaths. The proportion of patients who did not received RRT among patients with ESKD was greater in lower income countries. The largest differences in the number of patients with ESKD and those receiving RRT were observed in Asia, Africa and Latin America. Global use of RRT is estimated to increase up to 5.2 million over next two decades, with most growth in Asia. Conclusion: Globally, ESKD continues to cause many premature deaths, mainly in developing regions. The prevalence

of ESKD as well as RRT is projected to increase over next two decades, mainly in Asia, but a similar number of people will continue to die due to lack of access to treatment. Effective prevention and management of CKD, coupled with the development of affordable dialysis and kidney transplant services for ESKD should be priorities for the renal community. PERIYASAMY MUTHUKUMAR, THANIGACHALAM DINESHKUMAR, NATARAJAN GOPALAKRISHNAN, JEYACHANDRAN DHANAPRIYA, RAMANATHAN SAKTHIRAJAN, T BALASUBRAMANIAM Madras Medical College Introduction: Rheumatoid arthritis (RA), a chronic crippling disease can affect all Rapamycin components of the kidney. Renal involvement may be due to disease or drugs used to treat the condition. We intend to study the renal lesions in RA and its clinic o pathologic correlations. Methods: Prospective observational study Docetaxel ic50 was conducted at department of nephrology, Rajivgandhi Government general Hospital, Chennai, India between 2010 to 2013. RA patients with abnormal urine sediments (>3 RBC, s or RBC cast), proteinuria (>0.3 gms/day) or eGFR (<80 ml/min) were included in the study. Those with normal renal parameters were

excluded. Results: Three hundred patients with RA were screened. Mean follow up was 23 months. 52 patients found to have renal disease. Mean age was 45 years (range 18–67). 60 % patients were female. (Male: female ratio 1.5:1). Mean duration of illness was 8.5 years. 30% had odema, 4% had macrohaematuria, 52% were asymptomatic. The common renal syndromes observed in our study were chronic kidney disease (CKD-44%) Hypertension (20%), nephrotic syndrome(13%), acute kidney injury(4%). 29 patients (56%) underwent renal biopsy. The common histological pattern of renal biopsy observed were mesangial proliferation (10), focal endocapillary proliferation(5), IgA nephropathy(3), minimal change disease(2), membranous (2) and Amyloidosis(2).

As evidenced by outbreak investigations, the cutaneous commensal flora of the patient or health care workers is the usual source of the infecting organism.1,11,56,58 Apart from contamination during insertion or following administration of a contaminated parenteral solution, catheters may become infected by migration of organism from the exit site along the outer catheter wall or from the hub through the lumen of the catheter, adherence of the organism to the catheter material

with biofilm production, resulting in local replication and shedding of the organism in the blood.71,73–77 Microbial selleck screening library and host factors may play a role in localising the organisms to the catheter or in progression to fungaemia and clinical sepsis.62,78 However, even if host defences are able to clear the organism from the blood, the infection may not be resolved until the catheter is removed. Similar to catheter-related candidaemia, catheter-related Malassezia fungaemia has been associated with administration of parenteral lipid emulsions. While the exact mechanisms of this association remain unclear, it is conceivable that lipids administered through the catheter may provide a growth advantage for Selleck Idasanutlin Malassezia.56,58,76,79

On the other hand, parenterally administered lipids may negatively affect host immunity by blocking the reticuloendothelial system, reducing the generation of reactive oxygen species and decreasing phagocytosis by neutrophils in vitro and thereby contribute to clinical disease.73 The clinical signs and symptoms of Malassezia fungaemia and sepsis are generally non-specific. Depending on the severity of the infection, the most commonly reported symptoms in critically ill, premature infants have been fever and respiratory distress; other less frequent symptoms include lethargy,

bradycardia, hepatomegaly, splenomegaly, seizures and cyanosis.22,58,80 Respiratory distress may result in pneumonia or bronchopneumonia with an interstitial appearance on radiography. The main laboratory findings in this setting are leucocytosis or leucopenia, and thrombocytopenia. Affected patients usually are premature, low birth weight infants with multiple co-morbidities, extended hospitalisation, central venous catheters and parenteral nutrition including lipid emulsions.10,21,54,56,81,82 Catheter-associated Malassezia fungaemia is sporadic in immunocompromised the children and in adults and therefore clinical manifestations are not as well described as in infants. Fever appears to be universal;71 other symptoms and findings may include chills and rigours, myalgia, nausea and vomiting, respiratory distress with or without apnea, pneumonia, leucopenia, thrombocytosisis and less frequently, leucocytosis; signs of exit site inflammation are uncommon.2,12,59,71 Similar to the neonatal setting, the most common patient profile includes prolonged hospitalisation, the presence of central venous catheters and the use of intravenous fat emulsions.

brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus

originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that Selleckchem GSK1120212 the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis. “
“Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well

as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different JAK inhibitor enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis. “
“Here, a microdilution technique based on the M27-A2 protocol (NCCLS, 2002) was employed to compare the susceptibilities of Candida albicans

and Candida dubliniensis to essential oils extracted from plants used as spices. The chemical compositions of the essential oils were defined based on the analysis of retention indices obtained by gas chromatography–mass spectroscopy. Taken together, the results showed that the activity of the compounds against the two species was similar. “
“Summary  Telomeres are the nucleoprotein structures at the ends of linear chromosomes and NADPH-cytochrome-c2 reductase maintain the genomic integrity through multiple cell divisions. Telomeres protect the chromosome ends from degradation, end-to-end fusion and abnormal recombination and they also promote the end replication. The budding yeast Saccharomyces cerevisiae is the most well-studied model system with regard to telomere and telomerase regulation. Recently, the opportunistic fungal pathogen Candida albicans has emerged as an attractive model system for investigating telomere biology. Candida underwent rapid evolutionary divergence with respect to telomere sequences.

One microliter of EZ-Tn5 Tnp was mixed with 50 μL of the competent cells of YS-11. The mixture was placed in an ice-cold 2 mm-gapped cuvette (BioRad Laboratories Inc., Hercules, CA). The cells were transformed by electroporation

using Gene Pulser II (BioRad) at 2.5 kV, 25 μF, and 200 Ω. After electroporation, 1 mL of SOC medium (Invitrogen, Carlsbad, CA) was immediately added to the cell suspension, and the culture was incubated at 37 °C for 1 h. One hundred microliters of the cell suspension was plated on TSAY containing 50 μg mL−1 of kanamycin (Nacarai Tesque, Kyoto, Japan). Four hundred and eighty-six colonies grown on selection plates were transferred into TSBY containing 50 μg mL−1 of kanamycin 5-Fluoracil supplier for screening mutants deficient in exopolysaccharide production. The viscosity of spent culture media of 486 mutants was measured using a rotary viscometer (Tokimec Inc.) as described above. Mutants showing lower viscosity than that of the parent strain YS-11 were further investigated by means of SEM to observe selleck cell surface-associated structures as described previously. Mutants that had completely lost the meshwork-like structures around cells were selected as putative knockout mutants

for genes involved in the formation of biofilm-like structures. Southern hybridization was carried out to confirm a single insertion of transposon on genomic DNA. The genomic DNA from a mutant strain without exopolysaccharide production was purified using the GNOME Kit (Qbiogene Inc., Morgan Irvine, CA) and digested with a restriction enzyme PstI (Takara Bio, Ohtsu, Japan). The DNA fragments

Leukotriene-A4 hydrolase were electrophoresed on a 0.8% SeaKem agarose gel (Takara Bio), transferred to a positively charged nylon membrane (Hybond-N+, Amersham Biosciences Corp., Piscataway, NJ), and fixed on the membrane by UV light irradiation (HL-2000 HybriLinker, UVP Inc., Upland, CA). To detect an insertion of EZ-Tn5 Tnp, a digoxigenin (DIG)-labeled probe designed from the sequence of EZ-Tn5 Tnp was generated using the PCR DIG probe synthesis Kit (Roche Applied Science, Mannheim, Germany) with a primer pair (Table 1) to amplify a kanamycin-resistant gene in EZ-Tn5 Tnp (EZ-Tn5 Tnp sequence is available at http://www.epibio.com/pdftechlit/techlit_eztn.asp). The membrane was prehybridized (30 min, 65 °C) in a hybridization solution (DIG Easy Hyb Granules, Roche Applied Science) and subsequently hybridized overnight at 65 °C with 2 μL mL−1 of DIG-labeled probe in a hybridization solution. The detection of DIG-labeled probes was carried out according to the manufacturer’s instruction in a DIG Luminescent Detection Kit (Roche Applied Science). Alignments of flanking regions of the inserted EZ-Tn5 Tnp were analyzed using a DNA Walking SpeedUp Premix Kit (Seegene Inc., Seoul, Korea) according to the instruction of the kit.