In this study, we discuss the different molecular approaches for typing C. glabrata isolates. Recent advances in the use of molecular biology-based techniques have enabled investigators to develop typing systems with greater sensitivities. Several molecular genotypic approaches have

been developed for fast and accurate identification of C. glabrata in vitro. These techniques have been widely used to study diverse aspects such as nosocomial transmission. Molecular typing of C. glabrata could also provide information on strain variation, such as microvariation and microevolution. Idelalisib “
“Clinical diagnosis of invasive fungal infections (IFIs) is sometimes difficult, and obtaining an accurate assessment of trends concerning the prevalence of IFIs is a challenge. The aim of this study was to determine trends in the prevalence of IFIs from an autopsy survey. The retrospective review of autopsy records stored in Toho University was performed on all documented cases with fungal infection from 1955 to 2006. A total of 411 cases of IFIs were detected among 10 297 autopsies. The prevalence of candidiasis decreased from 3.6% (1981–93) to 2.0% Selleck RAD001 (1994–2006), and that of aspergillosis increased throughout the 52-year period and reached 2.0% (1994–2006). The prevalence of IFIs in the patient group comprising haematological disorders was significantly higher (19.9%) than in other patient groups (2.9%), of which the odds ratio was 18.4 for mucormycosis

and 10.0 for aspergillosis. The lung was the most common organ involved irrespective of major fungal species, and most cases with candidiasis showed multiple-organ infection. Results confirmed the increasing prevalence of aspergillosis and high risk of IFIs in the patient group with haematological disorders. IFIs

were also detected in an immunocompromised state caused not only by primary disease but also by treatment with anti-tumour drugs and corticosteroids. “
“There are discrepancies in the literature regarding the prevalence of tinea pedis in psoriasis. The aim of this investigation was to conduct a cross-sectional study of the prevalence of tinea pedis in psoriasis compared Adenosine to atopic dermatitis patients and normal controls. We enrolled 232 psoriatic patients, 190 atopic dermatitis patients and 202 normal controls, between the years 2010 and 2013. The prevalence of tinea pedis was 13.8% in psoriasis patients, not significantly different from that in atopic dermatitis patients 8.4% (P = 0.092)), but significantly higher than in normal controls 7.4% (P = 0.043). Both gender and age affected the prevalence of tinea pedis in psoriasis and normal controls, while only age affected the prevalence of tinea pedis in atopic dermatitis. Regarding gender, there was higher prevalence of tinea pedis in men: 19.1% (P = 0.019) in psoriasis and 12.1% (P = 0.013) in normal controls. Age affected the prevalence of tinea pedis in normal controls (P < 0.001), psoriasis patients (P = 0.

Importantly,

we found that proinflammatory cytokines may be dysregulated by a decreased STAT5. STAT5 normally stimulates an inflammatory response during bacterial infection [[36]]. Park et al. [[37]] have shown that Cav1 is a negative regulator of JAK2/STAT5a signaling in the mammary gland. This negative regulation may occur through direct molecular interaction owing to structural homology between Cav1 and SOCS-1 or SOCS3 [[38]]. Our data suggest that the GSK3β−β-catenin−Akt axis may be related PCI-32765 ic50 to a decreased STAT5 profile, making a connection from Cav1 deficiency to the exacerbated inflammatory response. Although the above research begins to hint at some important answers, it is not known why decreased STAT5 functionality leads to an increased proinflammatory cytokine profile. Previous reports have shown that Akt can connect

to STAT5 and regulate neuroprotective activity or cancer development [[39]]. However, little is known as to the specific functions of the GSK3β−β-catenin−Akt axis in bacterial infection. We hypothesized that decreased STAT5 may be regulated by changes in GSK3β or from the loss of Akt/β-catenin activity (at middle or late phases of infection), since our in vitro assays indicated an increase in pSTAT5 at early phases of infection. Following PIP3 and PI3K activation, Akt activation is required to regulate apoptosis against LPS or other oxidants [[40]], which could also be associated with a heightened inflammatory response. Akt is negatively regulated under Cav1 deficiency, while GSK3β is upregulated. Fostamatinib As feedback, Akt can inhibit GSK3β, thereby reducing the negative regulation of GSK3β in cellular processes. We assumed that an excessive inflammatory response and inefficient apoptotic clearance of dead cells lead to severe lung injury. Thus, an interaction between Akt and Cav1 may broadly impact the cytokine production and disease process.

Downregulation of Akt and STAT5 was initiated to counteract the loss of Cav1, but failed to eradicate the invading bugs. As a result, IL-6 and related cytokines could not be properly controlled by feedback signaling, Sinomenine contributing to the severe infection seen in cav1 KO mice. In summary, our studies illustrate a typical phenotype in cav1 KO mice following K. pneumoniae infection, characterized by increased bacterial burdens in the lung, decreased survival, severe lung injury, and increased inflammatory response. Furthermore, the increased impairment of the immune system in these KO mice is at least in part attributed to a regulatory function of the STAT5 pathway, which is, in turn, influenced by a GSK3β−β-catenin−Akt axis. Our studies have also characterized a novel role of Cav1 in infection resistance and explored its involvement with the Akt-STAT5 cross-talk, whose underlying mechanisms warrant further study. More specifically, our data may shed light on the pathogenesis of K. pneumoniae infection and suggest a novel therapeutic target.

Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour RXDX-106 cost cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency

and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness. Invariant NK T cells are a distinct set of T cells characterized by

expression of an invariant T cell receptor (TCR) Vα14-Jα18 chain, coupled preferentially to Vβ8·2,7 or -2 in mice or TCR Vα24-Jα18 and Vβ11 in humans [1]. NK T cells recognize glycolipids, rather than peptide antigens, presented by the major histocompatibility complex class I-like molecule CD1d. This results in rapid release of large amounts of T helper type 1 (Th1) [interferon (IFN)-γ] or Th2 [interleukin (IL)-4] cytokines, which in turn can activate dendritic cells, NK cells and B cells as well as conventional PLX4032 in vivo CD4+ and CD8+ T cells [2,3]. Thereby, NK T cells play a pivotal role as intermediates between the innate and the adaptive immune system and have the capacity to enhance host immunity to microbial infections and cancer as well as prevent autoimmunity [4–6]. In healthy individuals, the frequency of NK T cells in the peripheral blood is relatively low and ranges between 0·01% to 0·2% of total lymphocytes [7–9]. In cancer patients, NK T cell counts are reduced further compared to age- and gender-matched healthy controls [7,8] and usually defective in IFN-γ production upon stimulation [10,11]. Low circulating NK T cell numbers were found to predict poor clinical outcome in patients with Amobarbital head and neck cancer [12]. Attempts have been made

to stimulate NK T cell expansion with the glycolipid α-galactosylceramide (αGalCer) in order to stimulate anti-tumour responses in cancer patients [13–18]. In 10 of 17 non-small cell lung cancer patients this resulted in prolonged median survival time [19]. In an IFN-α trial of patients with metastatic renal cell carcinoma (RCC), a disease that has not been associated with high NK T cell numbers previously, we detected unusually high levels of circulating NK T cells in two of 14 patients. This prompted us to characterize these cells further to elucidate whether they were related to the therapy and had anti-tumour effectivity. All patients had primary metastatic RCC, patient B2 had clear cell RCC with sarcomatoid component and patient B7 had papillary RCC.

HHSN261200800001E and by the Department of Immunology, University of Washington. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services,

nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr. Dennis Klinman and members of his lab are co-inventors on a number of patents concerning CpG ODN and their use. All rights to these patents have been assigned to the Federal government. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, Venetoclax purchase but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supporting Figure 1. Level of IRF and NF-κB transcription factors in the cytoplasm of ‘K’ ODN stimulated CAL-1 cells. CAL-1 cells were incubated with 1 μM of ‘K’ ODN for the indicated times. Cytoplasmic lysates were extracted and analyzed by immunoblotting

for changes in the concentration of (A) various IRFs and (B) NF-κB p50 and p65. Lamin and a-tubulin were used to assess cytoplasmic purity and loading. Data are representative Selleck AUY-922 of 3 independent experiments. Supporting Figure 2: Effects of siRNA knockdown on mRNA expression. CAL 1 cells were transfected with siRNA to knockdown Sucrase gene expression. A, B, C) The knockdown efficacy of the indicated siRNA was evaluated by analyzing mRNA levels by RT PCR. Changes in mRNA levels (percentage indicated) were evaluated by comparison to cells transfected with control siRNA in each experiment. D, E) CAL-1 cells were transfected with siRNA but not treated with CpG ODN. Note that IL-6 mRNA levels were unchanged in these cells. Results were determined by

RT PCR with GAPDH used as the endogenous control. Data represent the mean ± SEM of 2-3 independent experiments experiments. Supporting Figure 3. Schematic representation of proposed role of IRF-5 and NF-κB in the induction of IFNß and IL-6 by ‘K’ ODN in human pDCs. “
“A decrease in the number of dendritic cells (DCs) is a major cause of post-sepsis immunosuppression and opportunistic infection and is closely associated with poor prognosis. Increasing the number of DCs to replenish their numbers post sepsis can improve the condition. This therapeutic approach could improve recovery after sepsis. Eighty C57BL/6 mice were subjected to sham or caecal ligation and puncture (CLP) surgery. Mice were divided into 4 groups: (1) Sham + vehicle, (2) Sham + DC, (3) CLP + vehicle, and (4) CLP + DC. Bone marrow-derived DCs (BMDCs) were administered at 6 h, 12 h, and 24 h after surgery.

37 RMA-S-Kd cells are H-2Kd-transfected TAP function-deficient lymphoma cells.38 RMA, RMA-S, and RMA-S-Kd cells proliferated in RPMI-1640/1 × medium for peptide–MHC class I binding experiments (Gibco Co. & Hyclone Co.). Transfected TAP-mutant cells are kind gifts from National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), USA. The RSV was multiplied in HEp-2 cells that were grown with minimum essential medium/1 × (Gibco Co. & Hyclone Co.). Virus was further titrated with plaque Sirolimus assay in HEp-2 cells. The RSV was obtained

from the American Type Culture Collection. As presented in the Supplementary material, Fig. S1, influenza A/WSN/33 virus39 was multiplied in MDCK cells cultivated

with Dulbecco’s modified Eagle’s minimal essential medium/1 × (Gibco Co. & Hyclone Co.). The quantification of the H1N1 A/WSN/33 virus was performed with a plaque assay in MDCK cells. Influenza A/WSN/33 virus was provided by Professor Betty A. Wu-Hsieh, which was purchased from the Department of Medical Biotechnology and Laboratory Science, Chang Kung University. The virus was cultivated in the USA. Influenza A/WSN/33 check details virus originated from the UK.39 The TAP function-deficient cells can distinguish peptides with higher affinity to MHC class I molecules from those with lower affinity. To detect the binding to different MHC class I alleles by variant peptides (Table 1) that are derived either from RSV or from influenza A virus, RMA-S and RMA-S-Kd cells were incubated with 10 μm of synthetic peptides at 37° following inducible expression of H-2 molecules at lower temperature. Comparison of peptide–MHC class I binding affinity between distinct variant peptides and the original M2:82–90, RMA-S-Kd

fantofarone cells were incubated with a serial dilution of M2:82–90 as well as with each variant peptide derived from M2:82–90 (Table 1) for measurement of the binding capacity of these peptides to H-2Kd molecules. The expression level of MHC class I molecules is presented as a shifted percentage of mean fluorescence intensity (MFI). The equation for calculation of the shifted percentage of MFI is as follows: Shifted percentage of MFI = [(MFIvariant– MFIcontrol)/(MFIoriginal– MFIcontrol) −1] × 100% where MFIoriginal is the MFI of the original epitope, MFIvariant is the MFI of variant epitopes and MFIcontrol is the MFI of the peptide without binding. BALB/c mice were infected with 105–106 plaque-forming units of RSV via the intranasal route. Two to three weeks following infection, spleen mononuclear cells from infected BALB/c mice were isolated to be re-stimulated in vitro with synthetic peptides derived from the RSV M2–1 protein sequence overnight for analysis of specific interferon-γ (IFN-γ) responses by the ELISPOT assay13 (BD Biosciences Co.). BALB/c mice were provided by the National Laboratory Animal Centre in Taiwan.

Donor site morbidity was evaluated using the Constant–Murley test for the shoulder unit. Follow-up ranged from 6 to 35 months (mean 20.6 months). Good or excellent results in mouth opening

and cosmesis were achieved in eight patients, speech was assessed as intelligible or normal in all but one patient and mean ambulation time after surgery was 2.5 days. Results of Constant score ranged from 45 to 70 (mean 60.6), and the main limitation encountered was elevation of the arm above the Torin 1 supplier head, which was seen in all but one patient confirming the low impact of the technique on the shoulder system. Low morbidity, early ambulation time, possibility of simultaneous harvesting with the tumor resection, large musculocutaneous paddles in the chimeric version of the flap are advantages of the STFF and makes it a good choice in elderly patients, when other bone containing free flaps are not indicated because of the related morbidity, when other flaps are not available or when wide composite defects are approached. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“In 1926, a physicist

at Harvard named William T. Bovie created an instrument, which revolutionized the medical profession—the unipolar electrocautery device. This incredible device could make surgical incisions and provide hemostasis as well. It came with a price, however, as it also created new risks and dangers in the operating room, such as electrical burns selleck and fires. To resolve some of these problems, a bipolar electrocautery device was developed. The historical development and principles of both unipolar and bipolar electrocautery will be discussed in this article. © 2010 Wiley-Liss, Inc. Microsurgery, 2010. “
“Acellular nerve allograft is a new option for bridging nerve Tyrosine-protein kinase BLK defects that allows appropriate diameter

matching. The aim of the study was to compare the histologic and functional recovery of nerve defects treated with acellular nerve allograft versus cabled sural nerve autograft. Fifty-four Sprague–Dawley rats were divided into one of three experimental groups. A unilateral 10 mm sciatic nerve defect was created and repaired with an acellular nerve allograft (Group A), three cabled sural nerve autografts in antidromic orientation (Group B), and the newly created segmental defect in antidromic orientation (reversed autograft) (Group C). Two rats in each group we evaluated histologically at 6 weeks while the rest of the groups were tested histologically and functionally at 12 weeks. There were no differences in histomorphometry between the groups at 6 weeks, but at 12 weeks at mid-graft there were differences. Group C had the highest fiber count which was statistically greater when compared to Group A (P = 0.023) and when compared to Group B (P = 0.001).

These data are in a full agreement with the observation that autoantibody-negative first-degree relatives exhibit proinflammatory Selleckchem Pirfenidone islet-specific T cell responses [14]. As T1D is a cell-mediated disease, the production of autoantibodies is considered to be an accompanying epiphenomenon. Unexpectedly, B lymphoid tyrosine kinase (BLK) was the top-scored immunorelevant gene when the DRLN group was compared to the control samples. Moreover, significant upregulation of genes related to humoral immune responses

such as CD19 and CD22 was also observed. Interestingly, BLK is also expressed in the pancreatic beta cells where it modulates their function [15]. Furthermore, an immunointervention approach based on B lymphocyte depletion resulted in deceleration of the severity associated with the progression of diabetes [16, 17]. However, the specific molecular mechanism(s) underpinning these observations is yet to be elucidated. Among other genes differentially expressed in the DRL group are members of Toll-like receptor family Panobinostat mw (TLRs) involved in non-specific immune responses. Notably, TLR6, TLR2 and their adaptor protein TIRAP (Toll-interleukin 1 receptor domain–containing protein) signalling the presence of evolutionary conserved bacterial structures. In this context, the upregulated status of TLR6, TLR2 and TIRAP is an unexpected finding because viruses rather than bacteria

are considered to be relevant to T1D development [18]. On the other hand, Dasu and Jialal [19] have reported that the amount of TLR2 and TLR4 ligands is significantly elevated in T1D, underscoring the proinflammatory nature of environment in which T1D develops [20]. Castiblanco et al. [21] described TIRAP S180L polymorphism as a common protective factor acting against the development of systemic lupus erythematosus; however, no association

with T1D has been reported so far. In this context, Reynolds and colleagues [22] recently reported that TLR2 signalling in CD4+ T cells promotes Th17 responses and regulates the pathogenesis of autoimmune disease. Thus, TLR signalling could be an important molecular link between innate and adaptive immune mechanisms involved in the pathogenesis of diabetes. As the hallmark of TLR activation is the production of proinflammatory cytokines Nintedanib (BIBF 1120) [23], the upregulated levels of these receptors could rather reflect their ‘default’ expression setting which significantly contributes to inappropriate inflammatory immunopathologies increasing the risk for the development of T1D. The importance of TLR genes in the pathogenesis of T1D is further strengthened by the fact that entire TLR-related signalling network is found to be differentially regulated. From other types of non-specific immune mechanisms, it is necessary to pinpoint the differences related to complement activity.

It has been shown previously that alternative proteolytic processing is possible for endogenously expressed cathelicidin peptides, which may lead to different physiological effects in vivo 37. Therefore, it is likely that the immunological response under investigation will be altered depending on the concentration, location, cell types, and the form of mCRAMP

released during the response. BGB324 order The role of AMPs in regulating the magnitude of the adaptive immune antibody responses has not been investigated extensively and the results to date are contradictory. LL-37 (20 μg/mL) was shown to decrease IgM and IgG2a production from mouse splenic B cells activated with LPS and IFN-γ, primarily through inhibition of cell activation and proliferation 16. In contrast, another study demonstrated that LL-37 (6 μg/mL) increased the sensitivity of human peripheral B cells to CpG, enhancing B-cell activation and increasing IgM and IgG production 14. Our data using mCRAMP (100 ng/mL) and purified mouse B cells agree with the latter study 14 and show that mCRAMP increases the amount of IgG1 and IgE Luminespib chemical structure antibody production in Camp−/− B cells. Of course, two obvious differences that may account for the discrepancies seen are the use of LL-37 versus mCRAMP peptides and mouse versus human B cells. In addition, another very important variable to consider is AMP concentration. Since

it is nearly impossible to measure the physiological concentration within the splenic microenvironment where these responses are occurring, we titrated the mCRAMP concentration within our culture system ranging from 1 ng/mL to 10 μg/mL. DOCK10 Consistent with previous findings 38, our data showed that mCRAMP at the highest concentration

tested induced cell apoptosis, while moderate concentrations increased IgG1 production, and the lowest concentration showed no effect on IgG1 production. These observations suggest that the AMP concentration within the microenviroment of an immune response may partially dictate the positive or negative effect on antibody production. Our in vitro and in vivo data show that T cells exposed to mCRAMP produce less IL-4. However, the possibility exists that other cell types are affected by mCRAMP and secondarily affecting the T cells. LL-37 has been shown to drive mouse DC differentiation and enhance IL-6 and IL-12 production, while inhibiting IL-4 production. In addition, LL-37-exposed DCs increased IFN-γ production from T cells and polarize them to Th1 cells 39. Our in vitro data clearly show that mCRAMP is capable of acting directly on purified T cells that were polarized to Th2 cells and decrease their IL-4 production. Similarly, our in vivo data show that T cells produced more IL-4 in the absence of mCRAMP expression. IL-4 is the critical cytokine for the IgG1 class switch, and its elevated expression in the Camp−/− spleen after secondary i.p.

Figure S3. Substantial differences between 2D and 3D kinetic parameters. (A) 2D affinity or (B) on-rate is plotted vs. their respective 3D counterparts [1] as log-log plots and fitted by linear regression with R2 Transmembrane Transporters activator and p

values indicated. (C) Comparison between 2D and 3D off-rates. The drastically different ranges of the parameter values in panels A and B, the drastically different off-rate values in panel C, and the low R2 values in panel B indicate substantial differences between the kinetic parameters measured in 2D vs. 3D. “n.a.” denotes “not available”. Figure S4. Example of a lifetime in thermal fluctuation assay. Panel A shows raw data of thermal fluctuation of bead position and panel B shows the corresponding sliding standard deviation (std.) of bead position. Bond association is signified by a sudden drop of position std. to below a threshold whereas dissociation by resumption of position std. to above the threshold. Figure S5. Hybridoma cells coexpressing TCR and CD8 show two-stage kinetics of binding to RBCs bearing gp209–2M:HLA-A2

complexes. (A-F) Experiments were conducted with micropipette adhesion frequency buy CX-4945 assay as shown in Fig. 3 but instead of CD8- lines, CD8+ cell lines were used. With the exception of W2C8, all the TCRs exhibit two-stage patterns in their binding curves. Surface densities of TCR, CD8, and pMHC are indicated. Each point represents mean ± SEM of Pa measured from 2–6 pairs of hybridomas cells and gp209–2M:HLA-A2 coupled RBCs. (G) Effective TCR–pMHC 2D affinities determined using CD8- cell lines match those determined from the first-stage binding using CD8+ cell lines except for W2C8. Effective 2D affinities of six individual TCRs when interacting with gp209–2M:HLA-A2

were measured using CD8- cell lines (open bar, replotted from Fig. 3C) and compared to those calculated from measurements using CD8+ cell lines (closed bar). The calculation is based on the assumption that the first stage of the adhesion Rucaparib cost frequency vs. contact time curve is mediated by the TCR–pMHC bimolecular interaction only. Error bars represent uncertainty based on error propagation from adhesion frequency measureme Figure S6. No Correlation between total dwell time (ta) and T cell function. (A) Kinetic parameters for the panel of TCRs determined by SPR [1] and the total dwell time (ta) calculated based on previous method by setting the rebinding threshold at 60,000/M.s [2]. (B) The correlation between the calculated ta values and Tcell function (IL-2 production). “
“The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration.

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 Pexidartinib 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 Pembrolizumab supplier 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

Depsipeptide nmr 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.