putida PCL1445 (data not shown). The stability of the plasmids and the transposon integration was

tested by subculturing in nonselective media (without antibiotic selection pressure) for approximately 30 generations. Samples of the subcultures were plated and colonies were screened for the expression of mcherry by fluorescence microscopy. Strain PCL1481 carrying miniTn7∷mcherry did not show any loss of integration. No loss of plasmid was observed for PCL1479 carrying pMP7604, whereas 3% of the colonies of strain PCL1480 carrying pMP7605 had lost fluorescence at day 3 (data not shown). A qualitative and quantitative analysis for mCherry production in P. putida PCL1445 tagged with pMP7604, pMP7605 and pMP7607 was performed in order to evaluate the resulting brightness of the different check details constructs. Cells of overnight cultures were visualized using fluorescence and light microscopy (Fig. 3a) and fluorescence was quantified using fluorometry (Fig. 2b). mcherry expression was detected at the single-cell level for all tagged strains. Microscopic and fluorometric analyses showed that strain PCL1480 (harboring pMP7605) produced the highest amount of mCherry and strain PCL1481 (containing miniTn7-mcherry)

produced the lowest amount (Fig. 3a and b). The strains PCL1479, PLX4032 PCL1480 and PCL1481 produced mCherry in a ratio of 15 : 95 : 1, respectively. No significant fluorescence was detected for P. putida PCL1445 cells and strains PCL1477 and PCL1478 containing the cloning vectors pME6031 and pBBR1MCS-5 (Fig. 2b). To evaluate the applicability of the mCherry marker vectors for tagging Gram-negative bacteria, several other Gram-negative spp., such as P. fluorescens WCS365 (an efficient root colonizer), P. aeruginosa PAO1 (a model strain for cystic fibrosis research)

and E. tarda FL6-60 (a fish pathogen and model for zebrafish immunology), were transformed with pMP7604 and pMP7605. This yielded PCL1700, PCL1701, PCA0241, PCA0242, PCA0239 and PCA0240, respectively. Fluorescence microscopy analysis showed the production of mCherry for all transformed strains (data not shown). Single colonies were isolated and overnight cultures were grown for quantitative analysis of mCherry production and comparison with P. putida PCL1445 (Fig. 4). Strains containing pMP7605 showed the highest mCherry OSBPL9 production. Comparable mCherry production levels were observed among the four strains tested, except for the one carrying pMP7605, which showed a lower level of expression in E. tarda FL6-60. To analyze the applicability of the mcherry-expressing constructs pMP7604, pMP7605 and pMP7607 in established test systems, which are not suitable for efficient application of antibiotic pressure, P. putida PCL1445-tagged strains were allowed to form biofilms on glass (in vitro biofilm assay) and on tomato roots (in vivo assay used to study root colonization). Using CLSM, the tagged strains were visualized at the single-cell level in both assays (Fig.

Clinical outcome was good for all patients after 15 days. The limitations of this study resided in its retrospective nature and the small number of cases. However, it may serve as a reminder to clinicians of epidemiological, clinical, and laboratory data associated

with this uncommon but potentially lethal disease. It also shows that the risk would appear to be significant in Africa and that lymphocytopenia is a common feature of leptospirosis. The authors state that they have no conflicts of interest. “
“Background. Imported diseases recorded in the European Union (EU) increasingly involve traveling immigrants returning from visits to their relatives and friends (VFR). Children of these immigrant families can represent a population of extreme vulnerability. Methods. A randomized cross-sectional ABT-263 purchase study of 698 traveling children under the age of 15 was performed. VFR traveling children and non-VFR (or tourist) children groups were compared. Results. A total of 698 individuals were analyzed: 354 males (50.7%) and 344 females (49.3%), with a median age (interquartile range) of 4 (2–9) years. Of these, 578 (82.8%)

had been born in the EU with 542 (77.7%) being considered as VFR, whereas 156 (22.3%) were considered tourists. VFR children were younger (4.7 vs 8.2 yr; p < 0.001), they had more frequently Tanespimycin been born in the EU (62.8% vs 20.1%; p < 0.01) and were more frequently lodged in 17-DMAG (Alvespimycin) HCl private homes (76.6% vs 3.2%: p < 0.001) and rural areas (23.2% vs 1.6%; p < 0.001). Furthermore, VFR remained abroad longer (51.6 vs 16.6 d; p < 0.001), the visit/travel time interval was shorter

(21.8 vs 32.2 d; p < 0.001) than tourists, and consultation was within 10 days prior to the departure (26.4% vs 2.7%; p < 0.001). The risk factor most differentiating VFR children from tourists was accommodation in a rural setting [odds ratio(OR) = 5.26;95%CI = 2.704–10.262;p < 0.001]. Conclusions. VFR traveling children showed a greater risk of exposure to infectious diseases compared with tourists. Immigrant families may represent a target group to prioritize international preventive activities. Despite an overall stagnation in arrivals since 2008, the European Union (EU) has remained the world’s largest destination during the 21st century.1 Tourism, international business travel, and migration make up this intense traffic, resulting in greater vulnerability to old, new, or re-emerging infectious diseases. Immigrants who have settled in the EU commonly travel to their native countries after having resided for long periods in the EU or other Western-style nations.2 Thus, a steady increase has been recorded in the cases of imported diseases among immigrants from the EU visiting friends and relatives (VFR immigrants).

ExsD acts as an antiactivator by directly binding to ExsA (McCaw et al., 2002). Consequently, the exsD mutant expresses the type III secretion regulon constitutively, even in the presence of calcium. ExsC functions as an anti-anti-activator by binding directly to and inhibiting ExsD selleck products (Dasgupta et al., 2004). Consequently, overexpression of ExsC results in a constitutive expression of the T3SS regulon, and deletion

of exsC renders the cell incapable of inducing type III secretion genes, even under low-calcium conditions. ExsE is a secreted substrate of T3SS and interacts with the anti-anti-activator, its cognate T3SS chaperone ExsC (Rietsch et al., 2005; Urbanowski et al., 2005). An exsE-null mutant constitutively expresses T3SS effector proteins such as exoU, exoS and exoT, whereas overexpression of ExsE prevents the induction of the regulon. Based on these studies, a simple model has been proposed for the association between transcription and secretory activity. Under high Ca2+ conditions, ExsE binding to anti-anti-activator ExsC

disrupts the complex between ExsC and ExsD, thereby allowing free ExsD to bind ExsA. In contrast, ExsE is released extracellularly following the activation of the type III secretion machinery at low Ca2+ concentrations. A decreased level of intracellular ExsE allows ExsC to sequester ExsD, Y-27632 supplier thus liberating ExsA, which then activates the transcription of the T3SS genes of P. aeruginosa (Yahr & Wolfgang, 2006). In the case of V. parahaemolyticus T3SS1, the genes for three proteins, VP1698, VP1699 and

VP1701, that share sequence similarities with the Pseudomonas ExsD, ExsA and ExsC, respectively, have been identified Bortezomib manufacturer (Fig. 1a). A previous study suggested that VP1698 and VP1699 are functionally orthologous to ExsD and ExsA, respectively, of Pseudomonas (Zhou et al., 2008). However, experimental evidence showing that VP1701 is a functional homologue of ExsC is lacking. Moreover, sequence annotation of the T3SS1 gene cluster of V. parahaemolyticus did not identify any CDSs predicted to encode homologues of ExsE. Thus, it is unclear whether a regulatory mechanism similar to that in P. aeruginosa is used by the T3SS1 system of V. parahaemolyticus. In this study, we identified vp1701 and vp1702 as functionally orthologous genes of exsC and exsE from P. aeruginosa and showed that T3SS1 gene expression is regulated in a fashion similar to that of the ExsACDE regulatory cascade of P. aeruginosa. Moreover, we demonstrated a role for H-NS in the negative regulation of the expression of the exsA gene. The V. parahaemolyticus strain RIMD2210633 (Makino et al., 2003) was used as the wild type (WT) in this study. The deletion mutants were constructed using a suicide vector, pYAK1 (R6Kori, sacB, cat), as reported previously (Kodama et al., 2002, 2007, 2008). The strains and plasmids used in this study are listed in Table 1. The primers used for plasmid construction are listed in Table 2.

Besides its central role in regulation of virulence, the agr locus has also been connected to β-lactam resistance possibly via regulation of autolysis (Piriz et al., 1996; Fujimoto & Bayles, 1998). The agr system consists of two adjacent transcriptional units, RNAII and RNAIII, which are transcribed in opposite directions from two divergent promoters, P2 and P3, respectively. RNAII encodes the precursor and the transporter of the autoinducing peptide, as well as a membrane-bound sensor that responds to the autoinducer Ku-0059436 manufacturer by activating the transcriptional

regulator AgrA. AgrA, which is also encoded by RNAII, in turn activates several promoters including P2 and P3 at the agr locus (Queck et al., 2008). RNAIII is the major effector of the agr system and regulates the expression of a large number of genes by base pairing with target mRNAs. This directly affects the expression of some virulence genes; for example, reduced translation of the spa mRNA encoding the cell surface-associated Protein A (Huntzinger et

al., 2005) and induced translation of the hla mRNA encoding the α-hemolysin (Novick et al., 1993; Morfeldt et al., 1995). However, the most widespread effect of RNAIII is probably caused by its inhibition of rot translation (Geisinger et al., 2006). Rot is a repressor of many genes encoding extracellular virulence factors like proteases and hemolysins, but also functions as a positive regulator of transcription (Said-Salim et al., 2003). Our previous model of reversal of methicillin resistance in MRSA by thioridazine only included the effect of the combinatorial treatment

on mecA, blaZ, XL184 cost and their regulators. However, we expect that treatment with thioridazine causes profound changes in gene expression as recently demonstrated in a clinical isolate of multidrug-resistant Mycobacterium tuberculosis (Dutta et al., 2010). Based on this, we find it likely that thioridazine in combination with oxacillin affects the expression or activity of additional proteins involved in the resistance mechanism besides PBP2a. In the present study, we sought to explore the possibility that additional genes besides mecA and blaZ are involved in the mechanism by which thioridazine resensitizes MRSA to oxacillin. We analyzed the expression levels of selected genes involved in Amisulpride β-lactam resistance and peptidoglycan biosynthesis in response to the combinatorial treatment. Furthermore, to assess the suitability of the treatment, we also tested the effect on selected toxicity and virulence/pathogenesis genes. MRSA ATCC strain 33591 was routinely grown at 37 °C with shaking in brain heart infusion medium (Difco) and Mueller–Hinton medium and agar (BBL) for subculture and maintenance. Minimal inhibitory concentrations for oxacillin and thioridazine are >256 and 16 mg L−1, respectively. MRSA cultures were grown to an OD600 nm of 0.

This behaviour was propagated downstream into the Pc promoter of the cat gene cluster, which responds to the benzoate-degradation intermediate cis,cis-muconate. The TOL plasmid thus imposes expression of the chromosomal Pb with a stochastic behaviour likely to result in biochemical heterogeneity of the otherwise genetically clonal population when exposed to benzoate as a growth substrate. “
“This report describes the inhibitory effect of pomegranate NVP-BEZ235 cost rind extract (PGRE) on the motility of uropathogenic Escherichia coli (UPEC), a common agent of uncomplicated urinary tract infections (UTIs). To this end, a fliC-lux reporter, as well as

Western blot analysis and scanning electron microscopy, was used to demonstrate that when UPEC strain CFT073 is exposed to PGRE, expression of the flagellin gene, fliC, and flagellin production decrease. In agreement with

these results, the swimming and swarming motilities of UPEC were observed to be hindered in the presence of PGRE. To evaluate the PLX4032 order effect of other pomegranate materials (PMs), the hydrolysable tannins in pomegranate (PG; punicalagin) and pomegranate fruit powder (PGP) were also investigated. Of the materials tested, PGRE had the strongest inhibitory effect on fliC expression and motility. Moreover, a fractionation of PGRE showed fractions with a molecular weight between 1000 and 3000 kDa to be the strongest inhibitors of fliC expression. check details Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract; we propose that PGRE might be therapeutically beneficial in the treatment and prevention of UTIs. Urinary tract infections (UTI) are the most commonly diagnosed disease in humans (Gupta et al., 1999; Gupta, 2002) with a majority of uncomplicated UTIs being caused by uropathogenic strains of Escherichia coli (UPEC) (Warren, 1996). Up to 95% of UTIs occur in an

ascending manner that begins with bacterial colonization of the periurethral area followed by infection of the bladder (Bacheller & Bernstein, 1997; Kaper et al., 2004). The uropathogens may then ascend the ureters to reach the kidneys and if left untreated, access the bloodstream and cause bacteremia. During UTI, the role of flagellum-mediated motility in the ascension of UPEC to the upper urinary tract and in its dissemination into the bloodstream as well as in the maintenance of persistent infection has been well established (Lane et al., 2005, 2007b; Wright et al., 2005). The bacterial flagellum is composed of a motor, a hook, and a filament (Berg & Anderson, 1973). Some bacterial species are able to move by rotating the filamentous portion of the flagellum, which is a polymer of flagellin subunits encoded by the fliC gene (Macnab, 1992; Chilcott & Hughes, 2000; Berg, 2003). Mutations in E.

Clinically, it is often difficult to differentiate between fungal and bacterial infections. Fungal keratitis is an infrequent cause of microbial Smad phosphorylation keratitis among contact lens wearers and may occur in 4% to 27% of such cases, depending on the type of lenses.12 A recent outbreak of Fusarium

keratitis in the United States has caused a recall of contact lens fluid by the FDA.13–15 Fungi frequently contaminate contact lens paraphernalia or the lens itself. The most frequently noted predisposing factor for fungal keratitis was improper lens care, which led to contamination of the contact lens treatment fluids and cases.16 In our case, the use of once-daily contact lens, as well as the negative cultures taken from the same batch of lens, makes such a possibility highly unlikely. The diagnosis of Fusarium keratitis should be suspected in every case of “soilborne” keratitis unresponsive to antibacterials. However, the ultimate way to reach a definitive diagnosis is by Sabouraud’s agar cultures and direct visualization of the fungi from corneal scrapings. Microscopic examination may mistakenly identify Oligomycin A supplier the case as aspergillosis, as occurred in our case,

because histopathology reveals acute-branching septate hyphae similar to those found in aspergillosis.5 A close collaboration is therefore needed between ophthalmology–pathology–microbiology and the infectious diseases team. Recent reports have proposed a role for confocal microscopy in the early diagnosis of infectious keratitis.17 Although confocal microscopy cannot show bacteria, it is Nutlin-3 solubility dmso useful in the identification of Acanthamoeba and fungal filaments. While cultures and smears remain the standard diagnostic methods for evaluating bacterial and fungal keratitis, they are lengthy processes and may take days and even weeks to obtain growth. Confocal microscopy offers a rapid in vivo visualization of the fungal filaments, allowing immediate initiation of treatment. However, reports of the use of this method remain anecdotal and at this time evidence is lacking to support

it as the only diagnostic method of fungal keratitis.18 Treatment consists of removal of the contaminated lens in addition to topical and probably systemic antifungal agents.19,20 Topical natamycin is the treatment of choice, given its excellent antifusarial activity in vitro, its corneal penetration, and its safety profile.21 We present the beneficial use of topical (and systemic) voriconazole in the treatment of such severe cases. Furthermore , in severe or recurrent cases of ocular fungal infections, systemic antifungal agents such as posaconazole, itraconazole, or voriconazole may be used.19 If therapy is delayed, fusarial keratitis may progress to endophthalmitis. Hence, rapid and accurate diagnosis of keratitis is essential if vision is to be preserved.

To reduce the rate of imported malaria, specific educational tools should be developed Entinostat datasheet for those at high risk to make them understand and become compliant with chemoprophylaxis. Malaria risk among travelers tends to decrease, but it remains a life-threatening risk at many destinations.1 Also in China,

the incidence rate of malaria decreased from 126.41/100,000 to 1.94/100,000 between 1950 and 2000, but morbidity has increased since the early 2000s mainly in two provinces, Yunnan and Hainan.2 Recently, malaria infections have been imported by Chinese international travelers from areas such as sub-Saharan Africa to provinces where malaria had been uncommon for many years.3–5 To evaluate the reasons for the increasing number of imported malaria buy Dabrafenib cases among returning Chinese travelers, we conducted an airport-based questionnaire survey in different geographic areas of the People’s Republic of China.

Similarly to other knowledge, attitudes, and practices (KAP) studies relating to malaria and travel health,6–8 our study was conducted from December 2009 to April 2010 in the departure lounges of five airports: the Guangzhou Baiyun International Airport, Guangdong province; the Capital International Airport, Beijing; the Pudong International Airport, Shanghai; the Qingdao International Airport, Shandong province; and the Nanjing International Airport, Jiangsu province. Health quarantine staff at these airports distributed questionnaires to Chinese international travelers over 16 years of age with destinations in malaria endemic and nonmalarious countries. These questionnaires were derived from the ones used in previous studies,9,10 and were translated into Chinese, tested for ease of comprehension with a limited number of travelers. Further adjustments were made to the questionnaire to accommodate for the different educational Progesterone backgrounds of our travelers. As travelers may visit destinations anywhere in the countries visited, only countries were evaluated in

this survey; the exact location within the country was not investigated in the questionnaire. We divided the total population into two groups, those with destinations in malaria risk countries and those in malaria-free countries (control group). Malaria risk destinations were defined according to the latest Centers for Disease Control and Prevention (CDC) “Yellow Book” also taking into account malaria-free areas within the destination countries.11 The high-risk endemic areas refer to all the countries that are listed “all areas with malaria” in the section “malaria risk information and prophylaxis, by country”; however, we labeled countries as low-risk endemic areas in which only parts are endemic for malaria. Nonmalarious areas refer to the countries that are marked with “none” in that list.11 The questionnaires were collected from the travelers before they boarded the plane. Data were entered into the Epidata 3.1 (Jens M. Lauritsen, Odense, Denmark) and analyzed with the SPSS 12.

Using PCR on these strains, we also found that short, 2.4 kb, and long, 2.7 kb, versions of both the MTT1 and the MAL31 genes are present (Dietvorst et al., 2005). We have extended these studies by cloning and sequencing long and short versions from two more lager strains and testing their ability to restore the growth of the A15 lager strain on maltotriose with antimycin A. Escherichia coli XL1-Blue (Bullock et al., 1987) was used for plasmid amplification. Standard methods were used for E. coli transformation (Inoue et al., 1990). Four lager

strains, A15 (Dr J. Londesborough, VTT Biotechnology, Finland), WS34/70 (Weihenstephan brewery, Freising, Germany), BS01 and BS07 (Heineken Supply Chain), were used in this study. Standard methods were used for yeast transformation (Gietz et al., 1995). Plasmid pRUL409 was constructed selleck products by inserting ARS1 from plasmid pNatCre (Steensma

& Ter Linde, 2001) into the XbaI and EcoRI sites of vector pHSS6 (Seifert et al., 1986), thereby introducing a NotI site next to the EcoRI site flanking the ARS. Plasmid pRUL409 was provided with a KanMX cassette (Wach et al., 1994) from pUG6 by inserting it as a BamHI–BglII fragment into its BamHI site yielding pRUL409(KanMX). This plasmid was digested with BamHI and learn more XbaI to clone the various MALx1 and MTT1 genes. To convert the resulting plasmids from a multicopy plasmid into a single copy plasmid, CEN4 from plasmid YCplac33 (Gietz & Sugino, 1988) was amplified using the primers CEN4SacI and CEN4EcoRV and inserted into the EcoRV and SacI sites of pRUL409(KanMX) containing one of the various

MALx1 and MTT1 genes. Yeast cells were grown in YP (Difco peptone 2%, Difco yeast extract 1%) containing 2%d(+)−glucose (Merck), maltose (Merck) or maltotriose (Fluka, 96% pure HPLC). When required, G418 (Duchefa) Tacrolimus (FK506) was added to the medium at a concentration of 200 μg mL−1, and antimycin A (Fluka) at a concentration of 3 mg L−1. Escherichia coli (XL1-Blue) was grown in Luria–Bertani broth and, if necessary, kanamycin was added to a concentration of 40 μg mL−1. For solid medium, 15 g L−1select agar (Gibco) was added to the liquid media. The primers used for PCR are listed in Table 1. PCR amplifications were performed with 50–100 ng genomic DNA. The amplification conditions were as follows: 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 5 °C below Tm of the primers and 1 min kb−1 to be amplified at 74 °C, followed by 10 min at 74 °C. The reactions were performed in a total volume of 50 μL and, per reaction, 0.5 μL Vent polymerase (New England BioLabs) was used with the buffer supplied. All PCR products were cloned into the pCR®-Blunt II-TOPO® vector (Invitrogen) before they were recloned into pRUL409(KanMX). For each of the four strains, 11 independent PCRs were performed. The sequences of the MTT1 and MAL31 gene isolates from strains BS01, BS07, WS34/70 and A15 cloned into vector pRUL409(KanMX) were determined commercially (Baseclear).

22q11 deletion syndrome (22q11DS) is one of the most common multiple anomaly syndromes, and many dentists are likely to meet patients with the syndrome. Odontological research has focused on describing and analysing conditions/concepts

based on the current state of knowledge within the dental profession. Yet, these research topics are not necessarily the most important issues for the patients. Aims.  To explore and describe, by use of Grounded theory, parents’ experiences of oral health issues and needs for dental care in their children with 22q11DS. Design.  Twelve parents from different regions in Sweden were interviewed. Analyses were carried out according to Grounded theory. Results.  Parents recognised good oral STI571 concentration health as important for the wellbeing of their children. Oral health was a concern and the parents described the fight for this as struggling in vain for good oral health in their child. Conclusions.  Parents not only described their children’s oral health as important but also hard to gain. Thus, it is important that all patients with disabilities, regardless of whether there is a defined medical diagnosis or not, are identified and Angiogenesis inhibitor well taken care of in the dental care system.

“
“International Journal of Paediatric Dentistry 2010; 20: 102–111 Purpose.  The purpose was to describe pathologic paediatric conditions associated with airway compromise adversely affecting dental treatment with sedation and general anaesthesia. Methods.  A review of available literature was completed, identifying pathologic paediatric conditions predisposing to airway compromise. Results.  Airway-related deaths are uncommon, but respiratory complication represents the greatest cause of morbidity and mortality during the administration of general anaesthesia. Differences in anatomy and physiology of the paediatric and adult airway

contribute to the child’s predisposition to rapid development of airway compromise and respiratory failure; juvenile rheumatoid arthritis, cervical spine injury, morbid obesity, and prematurity represent only a few conditions contributing to potential airway compromise of which the paediatric clinician needs to be aware. In all cases, thorough physical examination prior to treatment is mandated Niclosamide to affect a positive treatment outcome. Conclusions.  Successful management of children and adolescents with a compromised airway begins with identification of the problem through a detailed medical history and physical examination. Due to the likely fragile nature of many of these patients, and possibility of concomitant medical conditions affecting airway management, dental treatment needs necessitating pharmacological management are best treated in a controlled setting such as the operating room, where a patent airway can be maintained. “
“International Journal of Paediatric Dentistry 2010; 20: 366–373 Background.

Single crystals were obtained using a solution containing 20% (v/v) 2-propanol, 20% (w/v) polyethylene Glycol 4000 and 1.0 M Sodium Citrate pH 5.6. The crystals measured 0.30 × 0.25 × 0.15 mm after growing approximately one month at 291 K. X-ray diffraction data were collected using this website wavelength of 1.423 Å at a synchrotron-radiation source (MX2 beamline – Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil) using a MAR CCD imaging-plate detector (MAR Research™). The crystals submitted to X-ray diffraction experiments were held in appropriate nylon loops

and flash-cooled in a stream of nitrogen at 100 K without cryoprotectant. The best data set was collected with a crystal-to-detector distance of 75 mm and an oscillation range of 1° resulting in 104 images collected. The data were processed at 1.92 Å resolution click here using the HKL program package (Otwinowski and Minor, 1997) showing the crystals belong to P212121 space group and that they are isomorphous

to the crystals of MjTX-II complexed to stearic acid (Watanabe et al., 2005). X-ray diffraction data processing and refinement statistics are shown in Table 1. The crystal structure was solved by the Molecular Replacement Method using the program MOLREP (Vagin and Teplyakov, 1997) from CCP4 package v.6.1.13 (Potterton et al., 2004) and atomic coordinates of MjTX-II/stearic acid complex (monomer A with the stearic acid ligand omitted was used – PDB access code 1XXS) (Watanabe et al., 2005). Rounds of crystallographic refinement with CNS v.1.3 (Brunger et al., 1998) and manual modeling using the program Coot v.0.7 (Emsley and Cowtan, 2004) were used to improve the model, considering Rcryst and free R-factors. Polyethylene glycol (PEG) 4000, isopropanol and solvent molecules were added by CNS v.1.3 and Coot v.0.7 programs. Due to the lack of electron density in

some regions of the model, the following amino acid side chains were not modeled: monomer A – Lys16, Lys 36, Lys70, Glu86, Asn88 and Lys128; monomer B – Lys16, Lys57, Lys69, Lys70 and Lys128. The final model was checked in MolProbity program (http://molprobity.biochem.duke.edu/) (Chen et al., 2010). The coordinates were deposited in the MTMR9 Protein Data Bank with identification code 4KF3. Molecular comparisons of the structures were performed using the Coot v.0.7 program (Emsley and Cowtan, 2004) with only Cα coordinates. The structures of MjTX-II/stearic acid (PDB ID 1XXS) ( Watanabe et al., 2005), BaspTX-II (PDB ID 1CLP) in its native form ( Arni et al., 1995) and complexed to suramin (PDB ID 1Y4L) ( Murakami et al., 2005), BthTX-I (PDB ID 3HZD), BthTX-I/PEG4000 (PDB ID 3IQ3), BthTX-I/BPB (PDB ID 3HZW), PrTX-I/BPB (PDB ID 2OK9) ( Fernandes et al., 2010), BthTX-I/α – tocopherol (PDB ID 3CXI), PrTX-I (PDB ID 2Q2J), PrTX-I/α – tocopherol (PDB ID 3CYL) ( dos Santos et al., 2009), PrTX-I/Rosmarinic acid (PDB ID 3QNL) ( dos Santos et al., 2011a), PrTX-II/fatty acid (PDB ID 1QLL) ( Lee et al.