Eight focus groups (FGs) consisting of 5–9 MDT members were conducted (55 participants in total: 22 medical staff, 19 nurses and 14 pharmacists) in two hospitals who recently implemented electronic prescribing.

Participants were purposively sampled based on their use of the prescribing system and recruited using expression of interest forms via the MDT pharmacist. Each FG involved staff from an established MDT and included representation from all main users of the system: doctors, nurses, and pharmacists. The topics discussed MDTs’ experiences of how easy it was to AZD4547 ic50 use each prescribing system. FGs were taped, transcribed and content analysis undertaken. Institutional research ethics approval was obtained. Content analysis identified how the appearance of the prescription chart had changed; two sub-themes emerged. Legibility was raised by a number of FGs and was considered important in ensuring accurate review

and administration of medications. However, some participants Daporinad cell line highlighted that illegible handwriting could indicate prescriber uncertainty and would lead to more caution when reviewing and administering medicines. With the move to electronic prescribing, participants reflected that there were no subtle cues and the prescription was ‘quite convincing’, leading to greater, possibly false, confidence in the information than would have been the case with some hand-written prescriptions. The electronic prescription design was a concern as MDTs needed to view different screens to get the information they required: this made the prescription ‘story’ of what medications a patient had received, or would receive,

harder to comprehend. Navigating different screens, and remembering to do so during busy periods, created inefficiencies and additional implications to patient safety. All text appeared in the same colour and font in a specific list order but regular drugs rarely appeared on the first screen. Difficulty was encountered in deciphering and distinguishing each drug in order to ensure they were appropriate for the patient as ‘it all looks the same’. The amount of information displayed on each screen distracted from important information, ‘it no longer jumps out at you; you have to go looking Silibinin for it’. Electronic prescriptions had small displays that never filled the computer’s visual display unit; it was like trying to ‘run a hospital through a letter box’. Electronic systems were perceived as an improvement now that the prescription was ‘legible’. MDTs felt their ability to identify medication risks and get a clear picture (story) of what medications a patient was taking had been reduced due to the layout and intricacies of electronic prescribing. Information provided by the electronic prescription is not instantly clear compared to a paper prescription.

The rate of change in the proportion of LAC PPI, LAC statin and ibuprofen and naproxen usage and total dosulepin usage altered significantly following introduction of the NPI. The use of NPIs to influence primary care prescribing in Wales appeared to have varied results. The change in rate of use was significant for four of the nine indicators included in this study. Two of the four promoted medicines associated with a more favourable

risk-benefit profile (percentage ibuprofen and naproxen prescribing and dosulepin use), perhaps suggesting Obeticholic Acid that prescribers considered them to be of higher priority. The significant change in rate of LAC statin prescribing was contrary to the aim of this indicator. Although a non-significant prescribing rate change was apparent for the remaining five NPIs, it was possible that 12 months was not sufficient to observe a significant change and that a longer period check details of monitoring was required. Ongoing monitoring of these NPIs is the subject of further work.   Generic (%) LAC PPI (%) LAC statin (%) ACE (%) Ibu & Nap

(%) H&A (DDDs/1,000 patients) Dosulepin (DDDs/1,000 PUs) NSAIDs (DDDs/1,000 PUs) PPIs (DDDs/1,000 PUs) *p < 0.05; **p < 0.01 1. All Wales National Prescribing Indicators 2013–2014. All Wales Medicines Strategy Group. http://www.wales.nhs.uk/sites3/Documents/371/National%20Prescribing%20Indicators%202013-2014%20%5Bwebsite%5D.pdf Jose Manuel Serrano Santos, David Wright, Fiona Poland University of East Anglia, Norwich, Norfolk, UK This study aims to explore how Individualised Medication Administration Guides (I-MAGs) would be received and used in care homes for administering medication to patients with dysphagia (PWD). The implementation of I-MAGs could increase nurses’ clinical confidence. Pharmacist interventions in care homes could help standardise practice in medication administration. As conditions such as stroke, cancer, Parkinson's disease and Huntingdon's chorea are commonly found in care homes between 15% and 30% of

residents buy Staurosporine in care homes have been found to have difficulties in swallowing their medicines.(1) To address the difficulties associated with administering medicines to patients who cannot swallow (with dysphagia), Individualised Medication Administration Guides (I-MAGs) were introduced by a specialised pharmacist in Care for Elderly wards in a general hospital in East Anglia. The guides contained detailed information about how to administer each medication and they were individualised to the needs of the patient. The I-MAGs were printed in green forms and attached to the medication chart in order to be used in conjunction with it. The ward nurses reported an increase in their confidence when administering medication when I-MAGs were present in the ward.(2) Some patients with I-MAG were discharged to care homes where the I-MAG might have been equally useful.

The rate of change in the proportion of LAC PPI, LAC statin and ibuprofen and naproxen usage and total dosulepin usage altered significantly following introduction of the NPI. The use of NPIs to influence primary care prescribing in Wales appeared to have varied results. The change in rate of use was significant for four of the nine indicators included in this study. Two of the four promoted medicines associated with a more favourable

risk-benefit profile (percentage ibuprofen and naproxen prescribing and dosulepin use), perhaps suggesting GDC 0199 that prescribers considered them to be of higher priority. The significant change in rate of LAC statin prescribing was contrary to the aim of this indicator. Although a non-significant prescribing rate change was apparent for the remaining five NPIs, it was possible that 12 months was not sufficient to observe a significant change and that a longer period AZD1208 research buy of monitoring was required. Ongoing monitoring of these NPIs is the subject of further work.   Generic (%) LAC PPI (%) LAC statin (%) ACE (%) Ibu & Nap

(%) H&A (DDDs/1,000 patients) Dosulepin (DDDs/1,000 PUs) NSAIDs (DDDs/1,000 PUs) PPIs (DDDs/1,000 PUs) *p < 0.05; **p < 0.01 1. All Wales National Prescribing Indicators 2013–2014. All Wales Medicines Strategy Group. http://www.wales.nhs.uk/sites3/Documents/371/National%20Prescribing%20Indicators%202013-2014%20%5Bwebsite%5D.pdf Jose Manuel Serrano Santos, David Wright, Fiona Poland University of East Anglia, Norwich, Norfolk, UK This study aims to explore how Individualised Medication Administration Guides (I-MAGs) would be received and used in care homes for administering medication to patients with dysphagia (PWD). The implementation of I-MAGs could increase nurses’ clinical confidence. Pharmacist interventions in care homes could help standardise practice in medication administration. As conditions such as stroke, cancer, Parkinson's disease and Huntingdon's chorea are commonly found in care homes between 15% and 30% of

residents L-gulonolactone oxidase in care homes have been found to have difficulties in swallowing their medicines.(1) To address the difficulties associated with administering medicines to patients who cannot swallow (with dysphagia), Individualised Medication Administration Guides (I-MAGs) were introduced by a specialised pharmacist in Care for Elderly wards in a general hospital in East Anglia. The guides contained detailed information about how to administer each medication and they were individualised to the needs of the patient. The I-MAGs were printed in green forms and attached to the medication chart in order to be used in conjunction with it. The ward nurses reported an increase in their confidence when administering medication when I-MAGs were present in the ward.(2) Some patients with I-MAG were discharged to care homes where the I-MAG might have been equally useful.

The cell suspensions exhibited a time lag of several minutes before the fluorescence increases. Similarly, we described a lag in potassium efflux from Vero cells and GH4 cells using the same strain of B. cereus (NVH 75/95, Haug et al., 2010). Both the wild-type toxigenic NVH 75/95 culture supernatant and the NheC-deficient MHI 1672 strain with supplemented NheC yielded similar shaped responses. We interpret this delay to be

that necessary for the toxin to buy CH5424802 bind to the cells, oligomerize and form transmembrane pores. Propidium uptake in Vero cells was abolished when the Nhe was pre-exposed to DDM micelles. The addition of the mixture of culture supernatant and DDM to the cell suspension will dilute the DDM concentration such that Ferroptosis targets the micelles will disperse. Yet, because the toxin remains inactive, the Nhe component(s) binding of DDM micelles is a functionally irreversible process. This is consistent with the mechanism of pore formation by ClyA in which large conformational changes of the protein occur. ANS binding of the purified Nhe components indicates that NheB

exhibits the greatest changes in ANS fluorescence after exposure to DDM. Whilst the exact mechanisms underlying ANS binding to proteins remain undefined, changes in fluorescence have been widely used as a marker for conformational changes where exposure of hydrophobic regions of proteins favour increased binding and fluorescence. NheB was found to exhibit characteristic changes observed with another pore-forming toxin, namely increased fluorescence

intensity along with a ‘‘blue shift’’ in wavelength maximum (e.g. Sangha et al., 1999). We were unable to detect any evidence of ANS binding to NheA and the lack of increased fluorescence intensity with NheC suggested that DDM was exerting its effect predominantly through interaction with NheB. Whilst unhelpful as a measure of conformational change, intrinsic tryptophan fluorescence of NheB indicates that the three tryptophan residues ADP ribosylation factor are buried within the protein both before and after exposure to DDM. This is compatible with their position within the alpha helical bundle similar to ClyA where the fluorescence wavelength maximum does not change on exposure to DDM (Hunt et al., 2008). SEC experiments are consistent with DDM inducing oligomerization of NheB. The reason for the presence of two peaks at the elution time for monomeric NheB is not known. NheB was prepared from culture supernatants as it has proven difficult to express the protein recombinantly. Nevertheless, NheB yields a single band at 39 kDa after silver staining and immunoblotting. It is possible that the peak is an inactive breakdown product of NheB that lacks the epitope recognized by the monoclonal antibody.

meliloti 2011 grown in batch cultures at pH 6.1 displayed a significantly lower death rate during the subsequent acid shock compared with rhizobia that had been cultivated in batch at pH 7.0 (Fig. 1a). In these experiments, cells of S. meliloti 2011 were ZVADFMK grown to mid-exponential phase (OD600 nm=0.2) at pH 7.0 or 6.1 in Evans minimal medium and resuspended in an acid-shock medium (Evans, at pH 4.0; see Materials and methods). Rhizobia that had been precultivated in batch at pH 6.1 improved their decimal reduction time (D10) by a factor of 3.7 compared with rhizobia that had been grown in similar batch cultures under neutral conditions (a D10 of 16.6 h compared

with 4.3 h, respectively). In striking contrast, when parallel studies on survival at pH 4.0 were carried out with rhizobia harvested from the chemostat, no differences were observed between the cells collected at pH 6.1 and at pH 7.0 (Fig. 1b). Both types of rhizobia showed similar D10 values (c. 2.9 h), which Endocrinology antagonist were slightly lower than the D10 of the less-tolerant

rhizobia grown in the batch culture at pH 7.0 (Fig. 1a). We need to emphasize here that the dilution rates had been kept constant during the steady states reached at pH 6.1 and 7.0 in order to avoid changes in acid tolerance that could arise from differences in the duplication time of the rhizobia growing at the two pH. The results, thus, show that the ATR induced when rhizobia grow at low pH in batch culture cannot be triggered by the same acid pH under continuous cultivation, thus indicating that exposure to acidity per se is an insufficient condition for evoking a shift to the transient state of increased acid tolerance. In the previous section, we showed that cells collected from the continuous cultures have a comparable PRKD3 D10 upon subsequent severe acid shock, irrespective of the pH during cultivation. To evaluate how the same rhizobia compared in their symbiotic capabilities, we studied their nodulation kinetics after inoculation onto alfalfa plants growing

in Fåhraeus medium at pH 7.0 or 5.6. Nodulation of rhizobia from the neutral chemostat was better at pH 7.0 than at pH 5.6 (Fig. 2a), in agreement with previous results obtained with cells from batch cultures (Munns, 1970). The results from this experiment also indicated that bacteria grown in the chemostat at pH 6.1 nodulate with comparable kinetics at pH 7.0 and 5.6 (Fig. 2b, black vs. gray curves). That is, rhizobia grown at pH 6.1 did not significantly modify their nodulation kinetics when changing the pH of the plant medium. In addition, bacteria from the chemostat at pH 6.1 did not reach, at neutral pH, the same total number of nodules as the rhizobia grown at pH 7.0 (black curves, Fig. 2b vs. 2a). Overall, the results indicate that while bacteria grown in the chemostat at different pH did not significantly differ in their tolerance to a severe acid shock (Fig. 1b), they behaved differently when inoculated on M. sativa at pH 7.0 (Fig. 2a and b).

meliloti 2011 grown in batch cultures at pH 6.1 displayed a significantly lower death rate during the subsequent acid shock compared with rhizobia that had been cultivated in batch at pH 7.0 (Fig. 1a). In these experiments, cells of S. meliloti 2011 were selleck compound grown to mid-exponential phase (OD600 nm=0.2) at pH 7.0 or 6.1 in Evans minimal medium and resuspended in an acid-shock medium (Evans, at pH 4.0; see Materials and methods). Rhizobia that had been precultivated in batch at pH 6.1 improved their decimal reduction time (D10) by a factor of 3.7 compared with rhizobia that had been grown in similar batch cultures under neutral conditions (a D10 of 16.6 h compared

with 4.3 h, respectively). In striking contrast, when parallel studies on survival at pH 4.0 were carried out with rhizobia harvested from the chemostat, no differences were observed between the cells collected at pH 6.1 and at pH 7.0 (Fig. 1b). Both types of rhizobia showed similar D10 values (c. 2.9 h), which SB203580 cost were slightly lower than the D10 of the less-tolerant

rhizobia grown in the batch culture at pH 7.0 (Fig. 1a). We need to emphasize here that the dilution rates had been kept constant during the steady states reached at pH 6.1 and 7.0 in order to avoid changes in acid tolerance that could arise from differences in the duplication time of the rhizobia growing at the two pH. The results, thus, show that the ATR induced when rhizobia grow at low pH in batch culture cannot be triggered by the same acid pH under continuous cultivation, thus indicating that exposure to acidity per se is an insufficient condition for evoking a shift to the transient state of increased acid tolerance. In the previous section, we showed that cells collected from the continuous cultures have a comparable mafosfamide D10 upon subsequent severe acid shock, irrespective of the pH during cultivation. To evaluate how the same rhizobia compared in their symbiotic capabilities, we studied their nodulation kinetics after inoculation onto alfalfa plants growing

in Fåhraeus medium at pH 7.0 or 5.6. Nodulation of rhizobia from the neutral chemostat was better at pH 7.0 than at pH 5.6 (Fig. 2a), in agreement with previous results obtained with cells from batch cultures (Munns, 1970). The results from this experiment also indicated that bacteria grown in the chemostat at pH 6.1 nodulate with comparable kinetics at pH 7.0 and 5.6 (Fig. 2b, black vs. gray curves). That is, rhizobia grown at pH 6.1 did not significantly modify their nodulation kinetics when changing the pH of the plant medium. In addition, bacteria from the chemostat at pH 6.1 did not reach, at neutral pH, the same total number of nodules as the rhizobia grown at pH 7.0 (black curves, Fig. 2b vs. 2a). Overall, the results indicate that while bacteria grown in the chemostat at different pH did not significantly differ in their tolerance to a severe acid shock (Fig. 1b), they behaved differently when inoculated on M. sativa at pH 7.0 (Fig. 2a and b).

This study aimed to explore the potential implication in initial adherence or host specificity of the specific sequences. In the SSH library, we obtained 115 unique fragments that were specific to the bovine strain. These fragments include sequences with homology to genes or pathogenicity islands (PAIs) present only in other specific E. coli pathotypes

(e.g. VTEC) or other species (e.g. Klebsiella, Nitromonas), which are not known to be present in EHEC strains of serogroup O26. This heterogeneity supports the hypothesis of a horizontal acquisition of genomic regions from other pathogenic bacteria (Brzuszkiewicz et al., 2009; Juhas et al., 2009; Kelly et al., 2009). Moreover, it reflects the genomic plasticity of EHEC and/or E. coli strains. This finding supports the hypothesis learn more of Mokady et al. (2005), suggesting that this variation in the genome contents of E. coli could indicate that its evolutionary strategy tends to create a mixed assortment

of virulence factors coming from various pathogenic strains. This combination leads to a unique set of such factors, which helps the bacteria to better survive. The PAI ICL3 locus, first described by Shen et al. (2004) in the VTEC O113:H21 E. coli CL3, was found in 11.3% of the tested EHEC and EPEC strains of serogroup O26. These results are surprising when compared to those obtained by Girardeau et al. Ruxolitinib purchase (2009), suggesting that PAI ICL3 is unique to LEE-negative VTEC strains and that this locus thus provides a new marker for such strains. We have reported here that the locus could also be present in eae-positive strains belonging to a major serogroup involved in human diseases. Girardeau et al. (2009) have suggested that PAI ICL3 used to be present in most E. coli pathotypes but that many of these pathotypes have undergone extensive deletions [probably

via homologous recombination between insertion sequences (IS) elements, which removed almost the entire locus]. We can assume that our positive strains were not deleted for this locus. Another possible explanation is that these strains have recently Mannose-binding protein-associated serine protease acquired the PAI ICL3 locus via horizontal transfer, which hypothesis is supported by the fact that the PAI ICL3-positive strains are not closely related. Concerning host specificity, only one sequence appears to be statistically specific to human strains in comparison with bovine strains. Nevertheless, this sequence is only present in a few strains (7% of bovine strains and 33% of human strains) and therefore could not represent a host-specific marker. Moreover, three sequences were statistically associated with the pathotype (EHEC or EPEC), but these sequences were not present in more than half of the EPEC strains.

, 2009). In this work, we show that BTK inhibitor concentration the use of functional genes, as the bacterial LmPH gene, as a proxy to study microbial diversity of relevant microorganisms in leaf litter decomposition is possible. We are confident that the use of other functional genetic markers

of bacteria, and its extension to the study of fungi, will provide additional and interesting results to support the idea of changing microbial communities in the process of litter decomposition and increase our understanding of how microorganism interacts in ecosystem processes. The authors acknowledge the contribution of Anna Díez to laboratory work. This research was financially supported by the Spanish Government through projects CGL2009-08338 and CGL2011-30151-C02-01. “
“hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo).

A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global GSK2118436 cell line transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated

by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial leaf blight of rice (Swings et al., 1990; Niño-Liu et al., 2006). Like other Gram-negative phytopathogenic bacteria in the genera Erwinia, Pseudomonas, Ralstonia and Xanthomonas, Xoo possesses hypersensitive response and pathogenicity (hrp) genes, which play critical roles in conferring pathogenicity on host plants and triggering a hypersensitive response in nonhost plants (Alfano & Collmer, 1997). The hrp genes are involved in the construction Decitabine concentration of a type III secretion (T3S) apparatus, through which bacterial virulence-associated proteins (effectors) are directly delivered into plant cells (Büttner & Bonas, 2002). The expression of hrp genes is tightly regulated and is induced in planta, but suppressed in complex media. Appropriate hrp-inducing media have been established for several bacteria; the media are generally nutrient poor and likely to mimic plant conditions (Schulte & Bonas, 1992; Xiao et al., 1992; Wengelnik et al., 1996a; Brito et al., 1999; Tsuge et al., 2002).

Adverse effects have been reported with all antiretrovirals and are one of the most common reasons for discontinuation of treatment [2–4]. Some adverse events, such as gastrointestinal problems and hypersensitivity,

occur rapidly, within the first few months of starting treatment, while other adverse events, such as cardiovascular disease and pancreatitis, can take much longer to develop [5–7]. Such long-term adverse events can influence the durability of a regimen. Combination antiretroviral therapy (cART) EPZ015666 concentration regimens most often include a nonnucleoside reverse transcriptase inhibitor, such as efavirenz or nevirapine, or a ritonavir-boosted protease inhibitor, such as lopinavir [8,9]. cART regimens with durability as well as virological efficacy are required in order to achieve long-term virological

Atezolizumab mw suppression and to maintain CD4 cell counts at a level that significantly reduces the risk of morbidity and mortality. Many cohort studies have compared the short-term and long-term efficacies of different cART regimens [10–14], but less is known about the durability of different regimens, particularly in patients who have started a cART regimen more recently. If a regimen is virologically effective, durability can then be measured as the time to discontinuation of the regimen because of treatment failure or toxicity, or the rate at which changes occur

in potential markers of toxicity, such as liver transaminases and cholesterol. The aim of the study was therefore to compare the long-term durability of nevirapine-based cART regimens with those of efavirenz- or lopinavir-based cART regimens based on the time to discontinuation and the development of any serious clinical adverse events once virological suppression had been achieved and after at least 3 months on the drug to exclude discontinuations because of early-onset Carbohydrate potentially treatment-limiting toxicities that each of the three drugs may cause. The EuroSIDA study is a prospective, observational pan-European study of 16 599 HIV-1-infected patients from across Europe, Israel and Argentina. The study has been described in detail previously [15]. In brief, patients were enrolled into eight cohorts from May 1994. At each follow-up visit, details on all CD4 cell counts and HIV RNA measurements since the last follow-up visit are recorded as well as the date of starting or stopping any antiretroviral drug, the use of any prophylaxis against opportunistic infections, the date and type of development of any AIDS-defining illnesses, non-AIDS-defining illness or opportunistic infections, and death. Data are collected from the centres through follow-up forms at 6-monthly intervals and the database updated accordingly.

As a control, the cells Selleckchem RG7422 were suspended in 1 mM Tris–HCl (pH 7.2) at a low cell density (2000 cells mL−1) so that encystment could be avoided as much as possible. Aprotinin was purchased from Sigma-Aldrich, leupeptin and pepstatin from Peptide Institute Inc., phenylmethylsulfonyl fluoride (PMSF) from Boehringer–Mannheim, sodium fluoride (NaF) from Wako,

and sodium orthovanadate from Sigma. PMSF and pepstatin were dissolved in dimethyl sulfoxide (DMSO) to give 1 M and 1 mg mL−1 stock solutions, respectively, and the stock solutions were diluted 1000 times to produce solutions with final concentrations of 1 mM and 1 μg mL−1, respectively, and containing 0.1% DMSO. Leupeptin, aprotinin, and sodium orthovanadate were dissolved in pure water to give 1 mg mL−1, selleck inhibitor 1 mg mL−1, and 1 M stock solutions, respectively, and they were diluted 1000 times to produce final concentrations of 1 μg mL−1, 1 μg mL−1, and 1 mM solutions, respectively. NaF was dissolved in pure water to give a 200 mM stock solution for 200-times dilution to produce a final solution at 1 mM. All procedures were performed on ice or at 4 °C. The cells that had been stimulated to encyst for 1 h in a solution containing 1 mM Tris–HCl (pH 7.2) and 0.1 mM CaCl2 at a high cell density (50 000 cells mL−1) were collected by centrifugation (1500 g

for 2 min), rinsed in a macronuclei-isolation buffer [10 mM Tris–HCl (pH 7.2), 0.25 M sucrose, 3 mM CaCl2, and 10 mM MgCl2] (personal communication with Dr. Y. Kodama of Kochi University), and suspended in a buffer containing 0.2% Nonidet P-40 (NP-40). After being kept in this medium for 30–60 min, the cells were disrupted by pipetting; then, macronuclei were Megestrol Acetate collected by centrifugation (50 g for 5 min). To digest the sticky mucus-like materials that could cause co-adhesion of macronuclei, the pellets of the macronuclei were suspended in a macronuclei-isolation buffer (pH adjusted to 8.25 with NaOH) containing 10 mg mL−1 lysozyme (Sigma-Aldrich) for 1 h at 25 °C, followed by sedimentation of macronuclei by centrifugation (50 g for 5 min). For DAPI (4′, 6-diamidino-2-phenyl-indole

dihydrochloride) (Nacalai Tesque) staining, 2.5 μL of 0.02% DAPI (dissolved in pure water) was added to 0.5 mL of the suspension of macronuclei (final concentration of 0.0001%). After 5-min staining, the macronuclei were centrifuged (50 g for 5 min) and then suspended in a macronuclei-isolation buffer. The samples were observed under a fluorescence microscope (BX-50; Olympus) equipped with a WU filter set for DAPI staining. For immunofluorescence microscopy, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h and rinsed with PBST (PBS containing 0.05% Tween-20). The cells were suspended in 1% NP-40 in PBS for 1 h, and subsequently transferred into PBS containing 1% polyoxyethylene (20) cetyl ether (Brij 58).