It’s a bit like a diary’ (7) information included in the SPA need

It’s a bit like a diary’ (7) information included in the SPA needs to be endorsed by a trustworthy source, (8) SPA to include links for further advice including social networking facilities, ‘building something social into the app so kind of use the app to chat to other patients as well, share your feelings’ and (9) using the SPA would improve care and make patients feel more empowered, ‘it’s kind of empowering to be able to kind of log

your own process. The large volume of information given to patients has shown to be ineffective in dealing with patients’ information needs and ADR management, with patients feeling cut off from help once they are back at home. The use of an SPA is acceptable to patients for accessing information to manage ADRs as well as keeping in touch

with their healthcare team. These findings pave the way for the introduction of SPAs to support patients on oral chemotherapy with potential for pharmacists to take on the role of monitors, triaging alerts accordingly. 1. Nabhani-Gebara S, Kayyali R, Olszewska A. Patient Perception of Educational Materiel Surrounding their Cancer treatment. Eur J Oncol Nurs 2012; 16: S30. 2. Moretti F, van Vliet L, Bensing J, Deledda G, Mazzi M, Rimondini M,

Zimmermann C, Fletcher I. A standardized approach MG-132 in vivo to qualitative content analysis of focus group discussions from different countries. second Patinet Educ Couns 2011; 82: 420–428. Michelle King, Fiona Kelly, Sara McMillan, Adem Sav, Jennifer Whitty, Amanda Wheeler Griffith University, Gold Coast, Queensland, Australia Pharmacy staff know little about the roles and needs of carers despite them being regular clients of the pharmacy The burden of being a carer may result in sacrifices, including the health of the carer Carers want information and assistance that can help relieve their burden Pharmacy can support the health needs of carers, and provide information and signposting to relevant services Carers are regular clients of community pharmacy but are often overlooked as pharmacy staff focus on the prescriptions or needs of the person they care for. Unless carers divulge information about their role and how it affects them, pharmacy staff are often oblivious to what that role entails. This lack of awareness may mean that opportunities to ease the carer’s burden are missed.

cruzi arginine transport system, mostly studied during the last d

cruzi arginine transport system, mostly studied during the last decade. Genes of the TcAAAP family were amplified by PCR from gDNA and cloned into find more the yeast expression vector pDR196 (Rentsch et al., 1995). The following genes were chosen for the complementation assay: TcAAAP187 (Tc00.1047053510187.540), TcAAAP245 (Tc00.1047053510245.10), TcAAAP411 (Tc00.1047053511411.30), TcAAAP431 (Tc00.1047053510431.30), TcAAAP545 (Tc00.1047053511545.80), TcAAAP507 (Tc00.1047053510507.40), TcAAAP649 (Tc00.1047053511649.100), TcAAAP659 (Tc00.1047053507659.20), TcAAAP707 (Tc00.1047053508707.10), TcAAAP837 (Tc00.1047053503837.20) and TcAAAP069 (Tc00.1047053504069.120). Genes have been named

according to the organism T. cruzi (Tc), the transporter gene family (AAAP, TCDB 2.A.18) and the three last numbers of the systematic ID from the GeneDB. The Saccharomyces cerevisiae strain S288C (BY4742 MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0, can1∷kanMX4) was kindly provided by Dr Alejandro Colman Lerner (FBMC, UBA, Argentina). S288C Δcan was maintained on complete

yeast extract/peptone/dextrose medium. Ura+transformants were selected on synthetic complete (SC) medium, which is composed of 2% glucose, 0.17% yeast nitrogen base (without amino acids and ammonium sulphate), 0.5% ammonium sulphate, 0.1% histidine, 0.1% leucine, 0.1% lysine and 2% agar. For the recovery of canavanine-sensitive phenotype, 150 mg mL−1 of canavanine was added to the SC plates. Yeasts were transformed with pDR196-TcAAPs and an empty vector pDR196 according to Gietz & Woods (2002). Ammonium sulphate was added to media to reduce the background amino this website acid transport produced by general permeases (Courchesne & Magasanik, 1983). Saccharomyces cerevisiae transformants were grown in the media described above, harvested in the logarithmic growth phase and resuspended in phosphate-buffered saline (PBS) to a final OD600 nm of 1. To start the reaction, 100 μL of this cell suspension was added to 100 μL of PBS containing labelled l-[3H] arginine (0.1 μCi) at the indicated concentrations. Following incubation at the indicated times at 28 °C, PAK5 the reaction

was stopped by five volumes of cold PBS and centrifugation at 8000 g for 30 s; cells were washed twice with 1 mL of ice-cold PBS. Pellets were then resuspended in 0.2 mL of 1% SDS–0.2 N NaOH and counted for radioactivity in liquid scintillation cocktail (Packard Instrument Co., Meriden, CT). Differences in transport rates have been statistically analysed using a t-test and a cut-off P-value of 0.05. Sequences from the Tritryps genome projects were obtained at GeneDB ( and TcruziDB ( Assembly and analysis of the DNA sequence data, including prediction of ORFs, were carried out using the software package vector nti ver. 10.3.0 (Invitrogen) and the online version of blast at the NCBI (

communitypharmacyscotnhsuk/core_services/mashtml); frequency; frequency of use of the same pharmacy; most recent use of a pharmacy medicine;

most recent purchase of a pharmacy medicine and type of pharmacy normally used. Health status was measured using the question, Smoothened Agonist ‘in general would you say your health is’ with five response options (excellent, very good, good, fair, poor). The sample size was estimated to ensure inclusion of an adequate number of individuals who had purchased NPMs in the previous 2 weeks. A postal survey of 3000 individuals was estimated to generate 1350 usable forms (based upon a 50% response rate and a further 10% being returned by the post office unopened). It was estimated that 8% (n = 108)

of these respondents would have purchased a NPM[19] and 45% (n = 608) would have used a NPM in the previous two weeks.[20] Approximately 75% of consultations for NPMs are made as product requests, with the remaining 25% representing advice requests.[4, 7] It was estimated, therefore, that 25% (95% confidence Rucaparib interval (CI), 18.8 to 32.4) of consultations for NPMs would involve the provision of some (unprompted) information to MCAs. A minimum sample size of 104 + m (where m is the number of predictors) is suggested when testing individual predictors in regression models.[21] This reflects the standard approach to sample size calculations for TPB surveys. The predicted sample size of 1350 was therefore sufficient to examine the proposed predictors of self-reported behaviour, together with potential confounding factors such as consultation type and patient characteristics. Data were entered into SPSS (version 18) (PASW Statistics 18. SPSS Inc, Chicago, IL, USA). All TPB variables showed skewed distributions and thus medians (interquartile

ranges) are presented alongside from Cronbach’s alpha (Cα) to determine the internal reliability of the measures. Items with low internal consistency were removed. Univariate tests were used to investigate the relationship between demographic characteristics and ‘giving information’ (the behaviour) and BI (intention) as well as BI-WWHAM. The association between TPB variables and behaviour was explored first by Spearman rank correlations (rs) and then using logistic regression performed in three steps: step one explored the proximal predictors (i.e. those nearest to the behaviour: PBC and BI); step two added the distal predictors (i.e. those that operate via the proximal predictors: attitude and subjective norm) and step three added demographic and pharmacy behaviour variables that were related to behaviour. Linear regression was used to assess the relationship between TPB variables and BI to give information and BI-WWHAM.

5 to 4 mg/kg (Von Voigtlander & Moore, 1973; Akerud et al, 2001)

5 to 4 mg/kg (Von Voigtlander & Moore, 1973; Akerud et al., 2001) are much higher than those typically used in rats (0.05–0.25 mg/kg). The aim of the present study was to perform a more extensive morphological and behavioural

characterisation of the unilateral intranigral 6-OHDA AZD6738 price lesion mouse model and correlate the extent of damage to the mesostriatal DA projections with the magnitude of impairment seen in a battery of tests commonly used for assessment of motor impairments in rats. Based on this information we have devised a set of behavioural criteria that can be used to indentify well lesioned mice prior to any restorative or disease modifying intervention. In addition to the standard drug-induced rotation, cylinder and stepping tests, we have explored the usefulness of a novel sensorimotor integration test, the corridor task, originally developed for studies in rats (Dowd et al., 2005a), for quantification of behavioural impairments in 6-OHDA-lesioned mice. learn more A total of 129 female mice (Charles River; NMRI strain, weighing 25–35 g at the time of surgery)

were used in this study. 6-OHDA was injected unilaterally in the substantia nigra in 122 mice. The remaining seven mice served as intact controls. All mice were subjected to behavioural analysis in all tests described below and, based on the wide range of motor impairments seen in both drug-induced rotation tests and the corridor task, 40 of the 6-OHDA-lesioned mice were selected for further analysis in the present study, while the others were used in a different experiment. The selection was made so as to include animals that represented the full range of motor deficits induced by the intranigral 6-OHDA lesion, defined as mild, intermediate and severe impairments. In all behavioural tests the animals were numbered without any indication of treatment. The stability of the motor deficits over time was studied in seven

mice that exhibited severe behavioural deficits in the early post-lesion time-point. Beginning 6 weeks after lesioning, these mice were tested at regular intervals in the corridor, drug-induced rotation and stepping tests until Tacrolimus (FK506) 23 weeks post-lesion. In this experiment the seven unoperated mice were included in all tests as intact controls. The animals were housed under standard conditions with free access to food and water under standard 12-h light–dark regime (light 07.00–19.00 h). All procedures were conducted in accordance with guidelines set by the Ethical Committee for the use of laboratory animals at Lund University. 6-OHDA (Sigma, Sweden) was injected into the substantia nigra (SN) pars compacta under gaseous anaesthesia and analgesia (2% isoflurane in 2 : 1 oxygen/nitrous oxide), using a stereotaxic mouse frame (Stoelting Germany) and a 5-μL Hamilton syringe fitted with a fine glass capillary (external diameter 60–80 μm). The toxin was used at a concentration of 1.

In our previous

study, the S10-GERMS method was demonstra

In our previous

study, the S10-GERMS method was demonstrated to be a useful tool for bacterial classification at species, subspecies, and strain levels of genera Bacillus and Pseudomonas strains based on phylogenetic analysis (Hotta et al., 2010b, 2011). In this study, our research focused on the identification of diverse APEOn-degrading bacteria, the Sphingomonadaceae, in the environment using the S10-GERMS method. First, the ribosomal subunit protein masses were calculated by S10 and spc operon sequencing of the Sphingomonadaceae. To construct the database, their masses were corrected by the observed Selumetinib mass analyzed by MALDI-TOF MS. Finally, ribosomal subunit proteins as biomarkers coded in S10 and spc operons were selected based on the corrected database. The selected biomarkers enabled rapid bacterial identification and phylogenetic classification

of the Sphingomonadaceae selleckchem by constructing a ribosomal protein database. The Sphingomonadaceae strains listed in Table 1 were used in this study. A number of isolated APEOn-degrading bacteria were identified as diverse species of the Sphingomonadaceae in our laboratory. The NBRC and JCM strains were purchased from the National Institute of Technology and Evaluation (NITE)-Biological Resource Center (NBRC, Kisarazu, Japan) and the RIKEN BRC (JCM, Wako, Japan) through the National Bio-Resource Project of MEXT, Japan, respectively. Each bacterial strain was grown aerobically in the medium and at the temperature recommended by suppliers. Sphingopyxis macrogoltabidus Sphingopyxis terraea t-Octylphenol polyethoxylates (OPEOn), which have the commercial name Triton X-100 (TX-100), were purchased from Wako (Kyoto, Japan) and Aldrich Chemical Co., respectively. The liquid basal salt medium with 0.1% (w/v) TX-100 as the sole carbon source, named TX-A medium, Flavopiridol (Alvocidib) was used for APEOn-degrading bacteria. TX-A medium was described in our previous study (Hotta et al., 2010a). Chromosomal DNA was extracted from the bacteria

as described previously (Hotta et al., 2010b). The quantity and quality of the extracted DNA were estimated by measuring the UV absorption spectrum (BioSpce-mini; Shimadzu, Kyoto, Japan). PCR amplification of S10 and spc operons was performed using KOD containing dNTP at a concentration of 200 μM, each of the primers at a concentration of 4 μM, 100 ng template DNA, and 2.5 U KOD polymerase (Toyobo, Tokyo, Japan) in a total volume of 50 μL. PCR amplification conditions of S10 and spc operons were as follows: (1) 2 min at 98 °C, (2) 30 cycles of 10 s at 98 °C, 30 s at 50–55 °C, and 6.5 min at 68 °C. PCR and sequencing primers used in this study were designed on the basis of consensus nucleotide sequences of S10 and spc operons from seven genome-sequenced strains of the Sphingomonadaceae with the clustal x program for the alignment of nucleotide sequences (Table 2). The sequencing reaction was carried out using a bigdye ver. 3.

Thus far, the role of NE in odor processing in adult rats remains

Thus far, the role of NE in odor processing in adult rats remains less studied. We investigated the role of noradrenergic modulation in the MOB on odor detection and discrimination thresholds using behavioral and computational modeling approaches. Adult rats received bilateral MOB injections of vehicle, NE (0.1–1000 μm), noradrenergic receptor antagonists and NE + receptor antagonists combined. NE infusion improved odor detection

and discrimination as a function of NE and odor concentration. SB431542 solubility dmso The effect of NE on detection and discrimination magnitude at any given odor concentration varied in a non-linear function with respect to NE concentration. Receptor antagonist infusion demonstrated that α1 receptor activation is necessary for the modulatory effect of NE. Computational modeling showed that increases in the strength of α1 receptor activation leads to improved odor signal-to-noise ratio and spike synchronization in mitral cells that may underlie the behaviorally

observed decrease of detection and discrimination thresholds. Our results are the first to show that direct infusion of NE or noradrenergic receptor antagonists into a primary sensory network modulates sensory detection and discrimination thresholds at very low stimulus concentrations. ”
“In neonates, the stress of social isolation can alter developing neural circuits and Staurosporine ic50 cause mental illness. However, the molecular and cellular bases for these effects are poorly understood. Experience-driven synaptic AMPA receptor delivery is crucial for circuit organisation during development. In the Benzatropine rat, whisker experience drives the delivery of glutamate receptor subunit 4 (GluA4) but not glutamate receptor subunit 1 (GluA1) to layer 4–2/3 pyramidal synapses in the barrel cortex during postnatal day (P)8–10, whereas GluA1 but not GluA4 is delivered to these synapses during P12–14. We recently reported that early social isolation disrupts experience-driven GluA1 delivery to layer 4–2/3 pyramidal synapses during P12–14. Here, we report that neonatal isolation

affects even earlier stages of development by preventing experience-dependent synaptic GluA4 delivery. Thus, social isolation severely affects synaptic maturation throughout early postnatal development. ”
“Department of Molecular Microbiology, The John Innes Centre, Norwich, UK Cultech Ltd, Port Talbot, Neath Port Talbot, UK Phenazinomycin is a hybrid natural product consisting of two chemical entities, a phenazine and a cyclic terpenoid. Phenazinomycin exhibits potent activity against murine tumors and adriamycin-resistant P388 leukemia cells. Streptomyces iakyrus DSM 41873 is known to produce five actinomycin G2–G6. In the previous study, we identified the gene cluster directing the biosynthesis of actinomycin G2–G4. Inactivation of acmG5′ gene in the actinomycin G gene cluster in S.

FISH/CLSM allowed the discrimination between S sanguinis and P 

FISH/CLSM allowed the discrimination between S. sanguinis and P. gingivalis and determination of the relative proportions of all three species. A partially heterogeneous architecture of the biofilm, which may be due competitive binding, was observed. However, the distribution of the relative proportions of the three species in all experiments stayed unchanged. The heat flow at a given time (as determined using

IMC) was a measure of metabolic activities of all bacteria present, and it thus declines correspondingly if bacterial activity diminishes. Similarly, heat over time (i.e. the integral of the heat flow) is a proxy for the growth curve and approaches a maximum when metabolic activity decreases (Braissant et al., 2010). This metabolic decline and asymptotic biomass accumulation pattern is due to changes in the IMC ampoule internal see more environment that occur during bacterial metabolism; that is, exhaustion of available nutrients or electron acceptors or build up of metabolic waste products. The pattern of rise and decline of the metabolic activity of the biofilm was seen in the first 50 h (Fig. 3) exhibiting similarities in the behavior of the biofilm to common

Topoisomerase inhibitor liquid or solid culture studies (Braissant et al., 2010). Thus, cumulative heat correlates with cumulative bacterial biomass only during this early part when the biofilm still grows and until heat flow peak is reached. Once the heat flow has stabilized Ceramide glucosyltransferase at a constant level, the accumulation of heat is most probably not related to a net increase in bacterial numbers and production of fresh biomass, but, rather, to metabolic activities related to maintainance of the mature biofilm and survival of the present cells. Alternatively, it

can be hypothesized that during this steady state of the heat flow, growth rate is equal to bacterial death rate, resulting in a stable metabolically active bacterial population. This latter hypothesis is in line with the first one if equally low growth and death rates are considered. In the present study, between 72–480 h ca. 70% of the samples (n = 17) showed a low steady state heat flow comprised between 0.8 and 1.8 μW, whereas in the remaining 30% (n = 7), the values were found much higher (reaching from 8.6 to 86.0 μW). Assuming a heat flow of 2 pW per active bacterial cell (James, 1987), we calculated the number of active bacteria in the biofilm. This suggests that in the present samples showing the lowest steady state heat flow, ca. 4 × 105 to 9 × 105 bacteria remained active on the surface of the titanium disk (5 mm2), whereas this number is up to 4.3 × 107 in the samples having the highest heat flow. This result emphasizes major variability within biofilms that appear similar in microscopic analyses. On the other hand, the time required to reach the maximum heat flow showed only moderate specimen-to-specimen variability.

This analysis includes follow-up data to a median date of May 200

This analysis includes follow-up data to a median date of May 2009. Patients starting nevirapine, efavirenz or lopinavir together with exactly two nucleoside/nucleotide reverse transcriptase buy PD0325901 inhibitors (NRTIs) after 1 January 2000 were included in the analysis. Baseline was defined as either the date of first virological suppression (defined as a single viral load <500 HIV-1 RNA copies/mL) or 3 months after starting treatment,

whichever occurred later. Patients were excluded if they did not have a CD4 cell count or viral load measured in the 6 months prior to starting the new regimen or if they did not have any prospective follow-up. Treatment-experienced patients were included provided that they had not previously been exposed to any of the regimens of interest. Ethical approval for each participating centre is sought according to local regulations. Durability was measured as the rate of discontinuation of nevirapine, efavirenz or lopinavir, development of any serious non-AIDS-related adverse events, or worsening of other clinical or laboratory markers. The reasons for discontinuation were compared among the three regimens and the incidence of overall discontinuation

calculated. Time to discontinuation was determined using Kaplan–Meier methodology. Consistent with previous work [4,16] HM781-36B manufacturer in addition to discontinuation for any reason, analyses considered separately discontinuation because of toxicities or patient/physician choice and discontinuation because

of treatment failure. Reasons given for discontinuation were taken from patients’ notes and reported on standardized EuroSIDA follow-up forms (see forms at One reason for discontinuation per antiretroviral was collected. Discontinuation because of reported treatment failure included virological, immunological and clinical failure. Cox proportional hazards models, stratified by centre, were used to compare the risk of discontinuation among the three regimens. Patients many were followed until discontinuation of the main drug or their last recorded visit in EuroSIDA. Sensitivity analysis investigated discontinuation of any drug in the regimen and the durability of the three regimens in a subgroup of patients who were treatment naïve. The development of any serious non-AIDS clinical events or changes in clinical markers was compared among the three treatment groups using Poisson regression. Diagnosis of a non-AIDS clinical event was defined as the development of a non-AIDS-defining malignancy, pancreatitis, end-stage renal disease, grade III or IV hepatic encephalopathy, myocardial infarction, stroke or other cardiovascular disease. Changes in major clinical or laboratory markers were defined as developing or worsening anaemia, losing >10% of body weight at baseline, an increase in total cholesterol to >6.2 mmol/L or a decrease in high-density lipoprotein (HDL) cholesterol to <0.

Zumbrunn and colleagues, unpublished data) (Figure 1) For reason

Zumbrunn and colleagues, unpublished data) (Figure 1). For reasons of consistency, the experts assessed all risks separately for an average traveler

Acalabrutinib research buy to Africa, Latin America, and Asia/Pacific. The study was approved by the Ethics Committee of Basel. The visual psychometric measuring instrument applied to record the participants’ risk perception, pictorial representation of illness and self measure (PRISM), was developed in 1995 and validated for the assessment of the subjective burden of suffering in patients with chronic diseases.[15, 16] It consists of a white board in DINA4 format with a fixed yellow disc, symbolizing a subject’s “self” in her/his current life situation, and a movable disc, representing an illness, which is placed on the board by the subject (Figure 2). The distance between the “self” and the illness [self-illness separation (SIS)] is the primary outcome of PRISM and inversely proportional to the perceived importance of the illness. For this study, “life situation” was specified as the planned journey and “illness” replaced by nine

health risks. The primary outcome was adjusted to self-risk separation (SRS) as a proxy for the risk perception. According to the severity, frequency of occurrence, or estimated concern for travelers, the following risks were included: Erlotinib price general risk (overall danger of a specific journey), mosquitoes, malaria, rabies, MRIP epidemic outbreaks, sexually transmitted infections (STIs), accidents, terrorist attacks, and vaccination-associated adverse events (VAEs). In 2008, pre-travel data collection was carried out by a computer application of PRISM[17] (T. Zumbrunn and colleagues, unpublished data). For technical reasons, pre-travel data in 2009, expert data, and all post-travel data were collected using hard copies of the computer application. The

forms were scanned and distances measured by means of a computer-aided design (CAD) program.[18] The CAD coordinates were converted to the original scale (cm). Differences between the median perceptions of travelers and experts with nonoverlapping confidence intervals (CIs) were considered as statistically significant. The CIs were calculated using a bootstrap re-sampling method with 500 replicates. Linear regression was applied to detect differences among traveler subgroups and the SRS log10-transformed prior to analysis. A two-sided p value < 0.05 was considered as statistically significant. No adjustments for multiple testing were made. All analyses were performed using PASW Statistics 18 and R version 10.

Previously, we reported the presence of a bifunctional gene encod

Previously, we reported the presence of a bifunctional gene encoding spermidine synthase (Spe) and saccharopine dehydrogenase selleck kinase inhibitor (Sdh) in the Basidiomycota fungus

Ustilago maydis, confirming previous data from Cryptococcus neoformans (Kingsbury et al., 2004). This gene contains a 5′-region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3′-region encoding Sdh (Valdés-Santiago et al., 2009). Apparently, this chimeric gene is specific to Basidiomycota, because in a preliminary search, it could not be identified in several Ascomycota species. Spe catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Regarding lysine, it is known that fungi synthesize it via their exclusive mechanism, the α-aminoadipate pathway (see Xu et al., 2006). Sdh, also called saccharopine reductase, catalyzes the penultimate step in this pathway (Bhattacharjee, 1992). In the present work, we have performed an exhaustive analysis for the presence of a homologous gene in those Basidiomycota species whose

genome has been sequenced, in other fungal taxa, and in the rest of living organisms reported in data banks. With the results obtained and the experimental data of gene amplification by PCR in different species, we propose the use selleckchem of this gene as a molecular marker for Basidiomycota in general. Yarrowia lipolytica P01A was obtained from Claude Gaillardin (INRA), Saccharomyces cerevisiae S288C was obtained from American Type Culture

Collection (ATCC 26108), Mucor rouxii IM80 (ATCC 24905) was obtained from Salomón Bartnicki-Garcia (University of California, Riverside), Rhizopus oryzae 2672 was obtained from CECT (Colección Española de Cultivos Tipo), U. maydis FB2 was obtained from Flora Banuett (California State University, Long Beach), Coprinus cinereus UAMH4103 was obtained from University of Alberta Microfungus Collection and Herbarium, Ustilago hordei 8A was obtained Florfenicol from ATCC (90511); Ganoderma lucidum, Ganoderma sp., Schizophyllum commune, Pleurotus ostreatus, Rhizoctonia solani, Agaricus bisporus, Ustilago cynodontis, Tilletia foetida, and Bjerkandera adusta belong to the collection from Laboratorio de Micología (Universidad Autónoma de Nuevo León, Monterrey, NL, Mexico). Fungal strains were maintained in 50% glycerol at −70 °C. For propagation, strains were inoculated in liquid YPG medium [yeast extract (Difco), 2%; peptone (Difco), 1%, and glucose (Merck), 1%] and incubated at 28 °C for 18 h under shaking conditions (150 r.p.m.). Escherichia coli strain ElectroMAX™DH10B™ (Invitrogen Life Technologies) was used for transformation with the PCR-amplified products cloned previously in TOPO™4 vector (Invitrogen).