[21] Therefore, before applying the age-specific death rates to p

[21] Therefore, before applying the age-specific death rates to population in each age group, we converted the annual death rates to the 9-day–period death rate. To do so, we assumed that mortality rates in reference populations were constant throughout

the year. The Population Registration System is the source of population demographic data in Thailand. The Cabozantinib concentration system provides nationality status information including the authentication of birth and death certificates. The Civil Registration Act (No. 1) of B.E. 2534 and the additional revision (No. 2) of B.E. 2551 specifies that all deaths occur in Thailand must be registered within 24 hours of being witnessed. There is no specific death registration system for foreign nationals. The process of death reporting and registering is similar to the process for Thai citizens (Figure 1). In cases of unknown or uncertain death, the investigation officers are charged to investigate. As pursuant to Thailand Criminal Procedure Code 148, the investigative officials may conduct or request a forensic autopsy to determine the cause of death before issuing the investigation report to the next of kin. The next of kin is then required to submit the report to the local administration

office to obtain an authenticated death certificate. For deaths occurring MEK inhibitor within medical establishments, the attending physicians are authorized to issue the medical certificate of death. The original medical certificate of death is given to the next of kin, and a copy is kept in the hospital files. The next of kin is required to submit the medical death certificate to the local administration office to obtain an authenticated death certificate. All registered death records are automatically sent to the central database at the Bureau of Registration Administration,

Ministry of Interior. This database is shared with the Ministry of Public Health and the National Statistical Loperamide Office.[12-15, 22] As all authenticated death certificates are issued in the official Thai language, translated death certificates authorized by the embassy or general consulate are helpful for the next of kin in resolving assets and estate matters in their respective countries. The certification of death in Thailand classifies deaths into three categories: death within medical establishment due to medical illnesses; death outside medical establishment due to natural causes; and death due to unnatural or external causes such as suicides, homicides, deaths from beastly attacks, deaths from accidents, and deaths of unknown cause.[13, 14, 22] During the 17-month study period, between January 1, 2010 and May 31, 2011, there were a total of 1,295 deaths registered in the Chiang Mai Municipality. Of these 1,295 deaths, 102 (7.9%) were among non-Thai nationals, with 66 deaths registered in 2010 (64.

The enhanced performance described above for EuCl-OFX was also ob

The enhanced performance described above for EuCl-OFX was also observed against P. aeruginosa FQ-R2 (data not shown), exhibiting a bactericidal effect at sub-MIC ofloxacin concentrations in the early hours of the experiment. Eradication was achieved with EuCl-OFX at 2048 μg mL−1 (8 × MIC ofloxacin for

P. aeruginosa FQ-R2) within the first hour of assay. After brief exposure to EuCl-OFX, the zeta potential of P. aeruginosa FQ-R1 was modified in value and sign (from −26.8 to 14.5 mV). The cationic nature of Eudragit is the key factor contributing to its interaction with the negatively charged microbial cell surface. The binding neutralizes click here and even reverse the surface charge of the bacteria. At this stage, the change is reversible. Cultures under the action of OFX showed no effect, in agreement with that previously reported for Escherichia coli with ciprofloxacin (Dealler, 1991). Most of the cells treated with EuCl-OFX for 3 h revealed alterations in their shape, cytoplasmic density and irregularities in bacterial cell wall which could affect the functionality of PFT�� in vitro the normal cell membrane (Fig. 2a). Although ofloxacin-treated cells showed slight changes in cytoplasmic electrodensity (*, Fig. 2b), the bacterial membranes were still unaltered and cell morphology was preserved. Untreated controls show normal appearance (Fig. 2d). Exposure of P. aeruginosa

FQ-R1 to EuCl-OFX before adding detergent or lysozyme resulted in lysis of 5.6 ± 6.8% of cells (data selleck kinase inhibitor not shown). Similarly, treatment with polymyxin

B resulted in lysis of 8.5 ± 4.6% of cells. Bacteria culture was weakly sensitized by EuCl-OFX to Triton X-100 and lysozyme, but strongly sensitized to SDS (Table 2). Bacteria cell lysis by lytic agents following polymyxin treatment, a known OM-disorganizing agent, did not differ significantly. By contrast, cultures treated with ofloxacin did not differ with the control. DiBAC4 is fluorescent probe voltage sensitivity that enters depolarized cells (Müeller & Straüber, 2010), used to estimate damage of membrane potential in P. aeruginosa treated with EuCl-OFX. Figure 3 presents the effects of increasing concentrations of EuCl-OFX, drug-free polymer (EuCl) and free ofloxacin on the membrane potential for three isolates of P. aeruginosa. The negative controls showed the minimum relative fluorescence intensity (Fig. 3a, e and i). Accordingly, we considered the M1 range to be undamaged cells showing no significant depolarization of cytoplasmic membrane, and the M2 range to be damaged cells. The cell proportions exhibiting dye-associated fluorescence (M2) are expressed as percentages. The results indicate a rapid depolarization of cells treated with EuCl-OFX. After 1 h exposure, DiBAC4-associated fluorescence increases in intensity between 1 and 3 log orders, depending on the concentration and the strain analyzed.

[25] There is less evidence, however, regarding the effect of pat

[25] There is less evidence, however, regarding the effect of patient demographics on their own communication behaviour during healthcare consultations. It is noteworthy that in this current study, respondents who were married or living with a partner were less likely to give information although they did not differ

significantly on intention to give information. We can speculate that those living with someone Sunitinib manufacturer had already had the opportunity to discuss symptoms and reach a shared decision about an appropriate product to buy. Respondents with more education had less intention to give information, which might be due to them being more confident about their ability to find information to guide choice of product purchase. In this current study, intention to give information significantly predicted the behaviour, although one might expect there still to be some intention-behaviour gap as

intention did not fully explain behaviour. Other TPB variables worked through intention. Subjective norm, i.e. the belief that others think one should do the behaviour, was the strongest predictor of intention to give information for both intention measures. Previous research indicates that greater information exchange is associated with the purchase of more appropriate medicines and that this is likely to occur when the purchaser makes a non-product request, i.e. gives information about their health or needs, rather than requesting a specific selleck chemical product.[3, 11, 26] The current results suggest that interventions designed to encourage information giving in pharmacies,

including WWHAM information, should be directed at factors associated with subjective norm, e.g. what individuals think other people would like them to do. The analysis of specific beliefs suggests that it is the beliefs of the family, the person’s doctor and the NHS that matter. While it might be difficult to intervene to change perceptions of family beliefs or actual family beliefs, information giving might Janus kinase (JAK) be enhanced by interventions that persuade individuals that their doctor and the NHS think that giving information during consultations for NPMs was advisable. Because the evidence shows that a higher level of information giving to MCAs results in more guideline compliant NPM selling,[11] this would be a justifiable message to disseminate and one that is likely to be supported by medical and other NHS sources. Since attitudes towards information giving did not add significantly to predict the intention or behaviour, there would be little value in trying to persuade potential purchasers that the results of giving information might result in more favourable outcomes. Alternatively, interventions directly targeting the behaviour, e.g. by making the giving of information easier, might have a direct effect on BI and on behaviour.

Propensity score matching was performed using logistic regression

Propensity score matching was performed using logistic regression analyses, with the index treatment as the dependent variable and all measured baseline characteristics as independent variables. Covariates included demographics, indicators of disease severity and comorbidities; those that reached significance at the P ≤ 0.05 level were used to create the propensity score. For bDMARD compared to tDMARD use, propensity score covariates included age, chronic obstructive pulmonary disease (COPD)/asthma, diabetes, disease duration, number of tDMARDs, sex and steroid exposure. For within-bDMARD use comparisons (etanercept vs. adalimumab), propensity score covariates

included disease duration, number of tDMARDs and steroid exposure. Baseline characteristics included in this Screening Library order study were age (standardized to the end of the study period), sex, duration of disease (from first observed

RA diagnosis until the end of the study period), number of different tDMARDs prescribed, patient exposure to steroids (including betamethasone, cortisone, dexamethasone, fluocortolone, hydrocortisone, methylprednisolone, paramethasone, prednisolone, prednisone, prednylidene and triamcinolone), and comorbidities present in the 180-day period prior to initial RA diagnosis date, defined by ICD-9-CM codes. Comorbidities included diabetes mellitus, excluding type 1 (250.xx, excluding 250.x1 and 250.x3), COPD/asthma (493.xx), hypertension (401.xx) and hyperlipidemia (272.0, 272.1, 272.2 and 272.4). Cases were identified as any patient RO4929097 concentration CYTH4 with the presence of SBI requiring hospitalization or one or more ICD-9-CM codes for TB (010.xx–018.xx) or lymphoma (202.8) during the interval between the first RA diagnosis and study end. SBI ICD-9-CM codes included those for encephalitis (323.x, 054.3), endocarditis (421.x), meningitis (320.x, 049.x), osteomyelitis (730.0x, 730.1x, 730.2x), pneumonia (481.x, 482.x), pyelonephritis (590.x), septic arthritis (711.0x, excluding 711.08), and septicemia or bacteremia (038.x, 790.7). Exposure

to DMARD treatment was calculated in patient years, starting on the date of first RA diagnosis. For case patients, this included the number of years between the initiation date for tDMARD or bDMARD and the occurrence of the safety endpoint (SBI, TB or lymphoma). For non-case patients, this included the number of years between the first tDMARD or bDMARD initiation and the end of the observation period (31 December 2009). Only adverse events that occurred during the period of drug use or within 90 days following the last prescription administered were considered valid. In cases where multiple events occurred for one patient, all events were recorded. The incidence rate and incidence rate ratio (IRR) were computed for the propensity score-matched cohorts.

Cells were harvested at a middle logarithmic growth phase and was

Cells were harvested at a middle logarithmic growth phase and washed with phosphate-buffered saline. Bacterial cells see more and MnO2 particles were separated by a Percoll (GE Healthcare) density-gradient centrifugation according to a method described elsewhere (Page & Huyer, 1984). An iron

content of bacterial cells was determined according to a colorimetric method described elsewhere (Page, 1995) with modifications. Cells were suspended in 25 μL of 7% perchloric acid and extracted overnight at room temperature, followed by the extraction for 4 h at 90 °C. The extract was mixed with 5 μL of 0.1 M ascorbic acid, 140 μL of 2 mM ferrozine solution, and 30 μL of 0.1 M NaOH. An iron content was normalized to a total protein concentration determined using a Micro BCA protein-assay kit (Pierce). After Shewanella cells were grown in LMM under a MnO2-reducing condition, they were lysed in a detergent solution containing 5% (v/v) Triton X-100 and 50 mM HEPES (pH7.4). Cell lysates were subjected to a spectrometric assay to determine c-cyt contents (Myers & Myers, 1992). A content was estimated from a difference in absorbances of the α peak (at 552 nm) between dithionite-reduced and air-oxidized samples, and a specific content was estimated by normalizing a protein content. Shewanella cells were grown anaerobically in LMM under fumarate- or MnO2-reducing condition, and cells were harvested in exponential

log phases. RNA was extracted using a Trizol reagent (Invitrogen) and subsequently purified using an RNeasy Mini kit and RNase-Free selleckchem DNase set (Qiagen). RT-PCR and subsequent quantitative PCR were carried out using a LightCycler 1.5 instrument (Roche) with PCR primers listed in Table S1. Standard curves were drawn using dilutions of PCR fragments of target genes (omcA, mtrC, SO3032, and 16S rRNA gene). A specificity of the quantitative PCR was verified by dissociation-curve analyses. An mRNA level of a target gene (omcA, mtrC, or SO3032) was normalized to that of the 16S rRNA gene. After screening of approximately 5000 random Tn-insertion mutants, we obtained one mutant (N22-7) that

generated a smaller halo around its colony (the reduction of brown MnO2 to colorless Mn2+ resulted in the formation of a halo) than the wild-type selleck screening library MR-1 (WT). An ability of N22-7 to reduce MnO2 was also analyzed in liquid cultures and compared with that of WT (Fig. 1). Figure 1a presents appearances of 96-h cultures in the LMM/MnO2 liquid medium, showing that N22-7 was deficient in MnO2 reduction. Figure 1b shows time courses of MnO2 reduction in the liquid cultures (initial OD600 nm of 0.01), indicating that a MnO2-reduction rate of N22-7 (117 ± 15 μM h−1) was approximately half that of WT (230 ± 30 μM h−1). In contrast, when MnO2-reduction assays were initiated by inoculating with higher concentrations of cells (initial OD600 nm of 0.

The PCR mixtures for the MT Ivan mutants contained 6 mM Tris-HCl,

The PCR mixtures for the MT Ivan mutants contained 6 mM Tris-HCl, pH 8.5, 2.5 mM KCl, 1 mM 2-mercaptoethanol, 0.01% Triton X-100, 1.5 mM selleck products MgCl2, 100 ng DNA, 25 pmol of each primer, 200 μM dNTPs and 2.5 U Taq polymerase in a final volume of 25 μL. After an initial denaturation step of 2.5 min at 96 °C, 35 cycles of 1 min at 96 °C, 30 s at 55 °C (MT Ivan) or 50 °C (MT Iver) and 1 min at 72 °C were performed. After the last cycle, a final elongation step of 15 min at 72 °C was carried out. The purified fragments were used as templates in the second PCR step. The PCR mixtures for the second step were the same as those used for the first PCR, but without adding DNA or primers. After the addition of 50 ng of each fragment, the reaction was started with a first denaturation step of 2.5 min at 96 °C, which was followed by 20 cycles of 30 s at 96 °C and 20 s at 72 °C. Thereafter, the primers were HSP cancer added (MT Ivan primers 1 and 5, MT Iver primers 3 and 5, see Table 1). Following a first denaturation step of 30 s at 96 °C, 35 cycles of 20 s at 96 °C, 30 s at 55 °C (MT Ivan)

or 50 °C (MT Iver) and 2 min at 72 °C were performed. In a last step, 20 cycles of 30 s at 96 °C and 1 min at 72 °C are followed by a last elongation step of 15 min at 72 °C. The mutated genes and the vector pET11a were digested with NdeI and BamHI according to the manufacturer’s protocol (Fermentas, St. Leon-Rot, Germany) and ligated in 1 × ligation buffer and 1 U T4 ligase (1 h, 22 °C). Escherichia coli TB 1 (New England Biolabs, Frankfurt/Main, Germany) was transformed with the plasmids using heat shock. For the detection of clones that contain the insert, the plasmids were isolated using the GeneJET™ Plasmid Miniprep

Kit (Fermentas) and the DNA inserts were sequenced (GATC, Konstanz, Germany). Methyltransferase gene expression was performed in the E. coli expression strain BL21 (DE3) (New England Biolabs). The E. coli BL21 (DE3) cultures were grown in Luria–Bertani media containing 0.1 g L−1 Progesterone ampicillin and 0.1% glucose. The induction of MT Ivan C286A and MT Iver C277A was performed at 28 °C for 2 h with 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer. All the other mutants were induced at 18 °C for 16 h with 0.5 (MT Ivan D150A, MT Ivan D154A, MT Iver E88A, MT Iver C111A and C128A), 0.25 (MT Iver C210A) or 0.1 mM IPTG (all other mutants). After induction, the cells were harvested by centrifugation for 10 min at 10 000 g and 10 °C and stored at −20 °C. The cells were disrupted under aerobic conditions using a French pressure cell at 137 MPa and the cell debris was removed by centrifugation for 30 min at 10 000 g and 10 °C.

Prior study in S cerevisiae evolving under glucose-limited condi

Prior study in S. cerevisiae evolving under glucose-limited condition showed that in one evolving population, adaptive mutants from different lineages evolved similar mechanisms of adaptation based on both transcriptional and genotypic analyses (Kao & Sherlock, buy CX-5461 2008). Unfortunately, there exist few studies of time-course samples in C. albicans currently. In C. albicans, studies of in vitro isolates evolved in the presence of fluconazole found different replicate populations reached different fluconazole MIC levels, suggesting a divergence in resistance mechanisms between

different populations (Cowen et al., 2000). Further transcriptome studies of the same series of in vitro evolved isolates demonstrated similarities and divergences in potential resistance mechanisms between different lineages (Cowen et al., 2002); and while evidence seems to suggest that similar resistance mechanisms are present in isolates from the same population, because of the small number of time-course samples analysed, it is not clear whether there is convergence in resistance mechanisms among isolates within the same population.

During the emergence of drug resistance, each mutation that arises represents a step along the fitness landscape. An important question is whether, starting from the same point on the fitness landscape (same genotype), parallel populations will converge in their evolutionary trajectories (whether they will traverse

Y-27632 mw Digestive enzyme similar paths along the fitness landscape). Although no detailed studies exist currently to answer this question definitively, some prior experimental evidence suggests that early steps in the evolutionary trajectory may ‘influence’ the population down certain evolutionary paths. We will discuss some of the evidence here. First, similarities in gene expression profiles between several parallel populations were observed in transcriptome studies of the in vitro evolved populations by Cowen et al. (2002). Specifically, in two parallel populations they analysed, the transient changes in transcriptional expression profiles from time point isolates were very similar (Cowen et al., 2002), suggesting that convergence in evolutionary trajectories may occur. A study with parallel populations of S. cerevisiae subjected to either stepwise increases in or a single high concentration of fluconazole found similar mechanisms arising in independent populations under the same selection scheme (Anderson et al., 2003), suggesting that selection regimen may determine resistance mechanisms involved and that these resistance mechanisms possibly converge in parallel populations in S. cerevisiae. The other evidence comes from more detailed genotypic analysis of the same series of C. albicans isolates by Selmecki et al.

In nature, cyanobacteria experience diel light–dark (LD) cycles,

In nature, cyanobacteria experience diel light–dark (LD) cycles, which may exert significant effects on the phage life cycle. An investigation into the role of light revealed that cyanophage S-PM2 adsorption to Synechococcus sp. WH7803 was a light-dependent process. Phage adsorption assays were carried out under illumination at different wavelengths and also in the presence of photosynthesis inhibitors. Furthermore, phage adsorption was also assayed to LD-entrained cells at different points in the circadian cycle. Cyanophage

S-PM2 exhibited a considerably decreased adsorption rate under red light CDK inhibitor as compared with blue, green, yellow SCH772984 in vivo light or daylight. However, photosynthesis per se was not required for adsorption as inhibitors such as dichlorophenyldimethyl urea did not affect the process. Neither was S-PM2 adsorption influenced by the circadian rhythm of the host cells. The presence or absence of the photosynthetic reaction centre gene psbA in cyanophage genomes was not correlated

with the light-dependent phage adsorption. The cyanobacteria are unique among eubacteria in that the central feature of their metabolism is oxygenic photosynthesis. Unicellular cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the marine picoplankton and contribute significantly to primary production particularly in the oligotrophic regions of the oceans (Goericke & Welschmeyer, 1993; Li, 1995; Veldhuis et al., 1997). Cyanophages, viruses that infect these

cyanobacteria, are extremely abundant in the marine environment and were first Vildagliptin characterized in 1993 (Suttle & Chan, 1993; Waterbury & Valois, 1993; Wilson et al., 1993). The life cycle of a lytic phage following its release upon the lysis of an infected cell starts with a period of diffusive ‘search’ for a potential host, followed by adsorption, replication and the subsequent release of progeny. In the past, the study of phages was largely confined to those that infect heterotrophic hosts; however, the analysis of marine cyanophage–host interactions is revealing novel aspects of phage biology particularly with reference to the role of light. Light might be expected to influence any of these stages of the marine cyanophage life cycle. In the laboratory, research on cyanophage–host interactions is normally carried out under constant illumination; however, cyanobacteria in the natural environment are subject to a diel light–dark (LD) cycle. Therefore, it is important to know how cyanophage–host interactions might be affected by the shift from light to dark, which will help in the identification of the first marine cyanophage receptor.

“In Escherichia coli, cytosine DNA methylation is catalyze

“In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5′CCWGG3′. Although the presence of cytosine DNA methylation was reported over 35 years ago, the biological role of 5-methylcytosine in E. coli remains unclear. To gain insight into the role of cytosine DNA methylation in E. coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence

of the full-length dcm gene using the polymerase chain reaction; (2) examined the same H 89 order strains for the presence of 5-methylcytosine at 5′CCWGG3′ sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2′-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly

conserved nature of this DNA modification pathway. DNA bases are modified by postreplicative methylation by enzymes selleck compound termed DNA methyltransferases. In prokaryotes, the most common modified DNA bases are 6-methyladenine and 5-methylcytosine (5mC). The most recognized role of modified DNA bases is in restriction-modification (R-M) systems (Ishikawa et al., 2010). In each R-M system, there Etomidate is a restriction endonuclease that cleaves foreign DNA and a site-specific DNA methyltransferase that prevents cleavage of host DNA, and in some cases controls expression of the R-M system (O’Driscoll et al., 2005). However, some DNA methyltransferases are not found in conjunction with a cognate restriction enzyme and are termed solitary DNA methyltransferases. In addition to DNA adenine methyltransferase

(Dam), Escherichia coli possesses another solitary DNA methyltransferase termed Dcm for DNA cytosine methyltransferase (Marinus & Lobner-Olesen, 2009). The presence of Dcm was discovered in 1973 by Marinus & Morris. The dcm gene of E. coli K-12 contains 1419 base pairs, and the predicted protein is 472 amino acids (Bhagwat et al., 1986; Hanck et al., 1989). The protein contains ten conserved motifs and a catalytic cysteine residue that is found in all cytosine-5 DNA methyltransferases (Posfai et al., 1989). The Dcm protein methylates the internal C in the sequence 5′CCWGG3′ where W = A/T (Palmer & Marinus, 1994). 5mC is occasionally spontaneously deaminated in an existing C:G base pair, and a T:G mismatch is formed. The dcm gene is in an operon with the very short patch repair (vsr) gene and is controlled by the same promoter.

Bacterial strains and plasmids used in this study are listed in S

Bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. Photorhabdus luminescens TT01 was grown in Luria–Bertani (LB) medium at 28 °C, and strains of E. coli were grown in LB medium at 37 °C. Escherichia coli DH5α was used as the host R428 cell line for recombinant DNA cloning. Escherichia coli BL21 (DE3) was used as the host for expression of binary toxin genes. Plasmid pET28a (Novagen) was used as expression vector in BL21 (DE3). Plasmid pETDuet-1 (Novagen) was used as co-expression vector in BL21 (DE3). Total DNA was

extracted from P. luminescens TT01 using the alkali lysis method. It was used as template for amplification of plu1961 and plu1962 (GenBank accession no. BX571865). Oligos used in this study were listed in Table S2. Oligo pair Plu1961-F/Plu1961-R was used to amplify plu1961, and Plu1962-F/Plu1962-R was used to amplify plu1962. Both PCR products were double-digested by EcoRI/SalI and cloned into pET28a to generate plasmids pET-plu1961 and pET-plu1962, respectively. For co-expression of Plu1961 and Plu1962 in BL21 (DE3), Co1961-F/Co1961-R and Co1962-F/Co1962-R were used to amplify plu1961 and plu1962, respectively. PCR products of plu1961 and plu1962 were double-digested

by PstI/SalI and NdeI/XhoI, respectively, and cloned into pETDuet-1 sequentially to generate co-expression plasmid pET-pluBi. All the plasmids were confirmed by DNA sequencing. Plasmids BIRB 796 molecular weight pET-plu1961, pET-plu1962, and pET-pluBi were transformed into BL21 (DE3), and resultant strains were designated as BL21 (plu1961), BL21 (plu1962), and BL (Bi),

respectively. Recombinant strains were grown in LB medium with kanamycin (50 μg mL−1) or ampicillin (100 μg mL−1) at 37 °C to an OD600 between 0.6 and 0.8. Then, isopropyl-beta-d-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mmol L−1. After IPTG induction for 4 h, Montelukast Sodium aliquots of 1 mL bacteria culture were sampled and harvested by centrifugation (10 000 g, 1 min). Pellets were washed three times with distilled water and suspended in 0.1 mL lysis buffer (50 mmol L−1 NaH2PO4, 300 mmol L−1 NaCl, 10 mmol L−1 imidazole, pH 8.0). Then, cells were lysed by sonication and centrifuged at 10 000 g for 2 min. The supernatant was collected, and 10 μL aliquots were taken for SDS-PAGE. Soluble binary toxins (Plu1961 and Plu1962) were directly purified on 1-mL HisTrapTM HP prepacked columns (GE Healthcare), using an AKTA Purifier system (GE Healthcare; flow rate 1 mL min−1). The column was equilibrated in His A buffer (20 mmol L−1 sodium phosphate, 0.5 mol L−1 NaCl, 20 mmol L−1 imidazole, pH 7.4). Proteins were eluted using a step gradient up to 0.5 mol L−1 imidazole in His A buffer. Fractions were analyzed by SDS-PAGE, and the protein content of the pools was determined using the Bio-Rad Bradford reagent. The purified proteins were dialyzed against PBS buffer prior to application.