Fragment FP1 and its derivatives FP1-1 and FP1-2 (Fig. 2a) containing either the distal or the proximal half of the palindrome were used for EMSA. Fragment FP1-3, which contains 2 U of the distal half,

and fragment FP1-4, which contains 2 U of the proximal half of the palindrome in direct repeats separated by GGC, were also used (Fig. 2a). Results indicated that DNA fragments containing only the distal or the proximal half as well as those containing two copies of distal or proximal half of the sequence were not bound by the PhaR protein (Fig. 2b). The PhaR protein bound only to the DNA fragment containing the sequence in its native configuration. Thus, both halves of the palindrome are required for Palbociclib cell line formation of a stable PhaR–DNA complex, and the orientation of the motif is critical for efficient binding of the PhaR protein. To determine the nucleotides within the sequence −71TTCTGCGGCGCAGCA−57 that are required for PhaR binding, various deletions including the T residue at position −71 and A at position −57 (Fig. 2a, FP1-5), both Ts at positions −70 and −71, and both the C residue at position −58 and the A residue at positions −57 (Fig. 2a, FP1-6), were performed. None of these deletions were found to have any effect on PhaR

binding (Fig. 2b). However, deletion of the first GSK2118436 nmr three nucleotides from both ends (Fig. 2a, FP1-7) abolished PhaR binding. Therefore, the PhaR-binding sequence was narrowed down to the 11-bp CTGCGGCGCAG symmetrical palindrome. Because the PhaR-binding sequence identified in this study is novel, careful analyses were performed to determine the importance of each nucleotide in the sequence for PhaR binding. A series of base substitutions were generated in either half of the CTGCGGCGCAG motif, including changing the first four bases from CTGC to ATGC, CAGC, CTAC, or CTGA and the last four bases from GCAG to GCAT, GCTG, GTAG, or TCAG (Fig. 2a, FP1-8, FP1-9, FP1-10, and FP1-11). All of these mutations were found to abolish PhaR binding (Fig.

2b). To investigate the importance of the three spacer nucleotides GGC in PhaR binding, the first G (Fig. 2a, FP1-18), both Gs (Fig. 2a, FP1-19), or the entire GGC (Fig. 2a, FP1-20) were deleted. Calpain All of these deletions abolished PhaR binding. Thus, the 3-bp spacing between the two halves of the palindrome is important for PhaR binding. To determine whether the spacer region must be GGC, it was replaced by GGT (Fig. 2a, FP1-12), GGA (Fig. 2a, FP1-13), GGG (Fig. 2a, FP1-14), CAT (Fig. 2a, FP1-15), ATG (Fig. 2a, FP1-16), or AGCC (Fig. 2a, LM17), and all of these mutations were found to have no effect on PhaR binding (Fig. 2b). These results indicated that PhaR recognizes a specific sequence, but the spacer region can be any three or four nucleotides.

In general, parents tend to estimate the dental fear of their children slightly higher than their children. “
“International Journal of Paediatric Dentistry 2010; 20: 305–312 BAY 80-6946 cell line Background.  Kallmann syndrome (KS) is a rare genetic disorder characterised

by central hypogonadism with a lack of sense of smell and in some cases renal aplasia, deafness, syndactyly, cleft lip/palate, and dental agenesis. To date, five genes for KS have been identified: KAL1, located on the X chromosome, and FGFR1, PROKR2, PROK2 and FGF8, which are involved in autosomally transmitted forms of KS. Aim.  The study characterised the dental ageneses of individuals with KS associated with mutations in the FGFR1 gene. Design.  Six individuals displaying dental agenesis were included. Clinical and radiological dental evaluations as well as

medical anamneses were carried out. Results.  Microdontia, screwdriver-shaped mandibular incisors, thin molar roots, and patterns of dental agenesis in both dentitions were observed. One to nine teeth were missing, most frequently, in descending order, lateral mandibular incisors, second premolars of upper and lower jaws, and lateral maxillary incisors. The pattern of dental agenesis is associated with four new mutations in the FGFR1 gene. Conclusion: Dental agenesis may be a clinical feature of Kallmann syndrome caused by a mutation in the FGFR1 gene. These findings highlight the role that odontologists selleck can play in the early diagnosis and treatment of gonadotropic deficiency. “
“Knowledge of the genetic and environmental influences in caries aetiology has relevance for preventive Sodium butyrate dentistry. This classical twin study compared concordance of mutans streptococci (MS) and lactobacilli (LB) colonization, enamel defects, and caries in a cohort

of 4–6-year-old mono- (MZ) and dizygotic (DZ) twin pairs. The twins were examined for prevalence and concordance of enamel opacities and hypoplasia, oral counts of MS and LB, and dental caries. Bacterial counts were assessed using a commercial microbiological kit. Thirty-four MZ and 50 DZ twins (mean gestational age 35.0 ± 2.4 weeks, and birthweight 2.4 ± 0.6 kg) were examined. There were no statistically significant differences between MZ and DZ twins in the prevalence of MS, LB, and enamel hypoplasia. Concordance rates for MS and LB presence and prevalence of enamel defects within MZ and DZ twin pairs were not significantly different. There were more children with caries in DZ compared with MZ twins (18% vs 3%, P = 0.0029), most likely due to increased daily frequency of sugar consumption and less toothbrushing. Concordance data from MZ and DZ twins did not demonstrate any statistically significant difference in susceptibility for enamel defects and colonization of MS and LB. “
“Facial and dental appearance influences how individuals are perceived by others. This study aimed to determine whether young people make judgements about other young people with visible enamel opacities.

These short-chain carbon molecules have also been reported to have inhibitory properties against S. cerevisiae and C. albicans (Bergsson et al., 2001; Kubo et al., 2003). The size of the chain length is clearly an important factor, which was confirmed in studies of the activity of 40 isomers of farnesol, which concluded that subtle changes in the structure of farnesol can have dramatic effects on the activity against C. albicans (Shchepin et al., 2003). At the molecular level, it is likely that these molecules act to influence key transcription factors, leading

to hyphal repression. Both farnesol and dodecanol were shown to affect the cAMP-controlled Ras1-Cdc35 pathway, which is integral to filamentation (Davis-Hanna et al., 2008). Genome analysis of Aspergillus species indicates that Rucaparib mouse cAMP signalling is conserved, thus indicating that these small 10 carbon molecules may play a pivotal role in fungal population control (Lafon et al., 2006). Moreover, recent transcriptional studies to examine the effects of P. Venetoclax research buy aeruginosa supernatant on C. albicans biofilm formation demonstrated that 236 genes were differentially

regulated, and interestingly, genes involved in adhesion and biofilm formation were downregulated, in particular YHP1, which encodes a protein known to inhibit biofilm formation (Holcombe et al., 2010). The suppression of other microbial pathogens via the secretion crotamiton of small molecules may play a pivotal role in microbial competition. Within the environment of the CF lung, bacteria and fungi

exist within close proximity, and given that bacterial quorum-sensing molecules have been identified directly from sputum samples of CF patients, it is plausible that complex microbial interactions are modulated through small defined chemical messengers to allow different bacteria and cross-kingdom interactions to take place that impact microbial pathogenicity (Singh et al., 2000; Shirtliff et al., 2009). Investigation of P. aeruginosa clinical isolates from CF patients has shown that the genetic diversity of quorum-sensing networks is common, with 19 out of 30 CF patients reported to contain lasR mutants (Smith et al., 2006). This indicates that P. aeruginosa may evolve within the complex microbial environment to allow its coexistence with eukaryotes, which is supported by data from a recent study describing mutual inhibition (Bandara et al., 2010b). Interesting observations from the same group showed that exogenous lipopolysaccharide was able to inhibit and disrupt Candida spp. biofilms in a time- and concentration-dependent manner (Bandara et al., 2010a). Collectively, the data demonstrate that P. aeruginosa has the ability to modulate C. albicans behaviour in a number of ways, and under certain circumstances, it can mutually coexist.

Double bands were selected only when two distinct bands could be seen on the gel image and in the bionumerics densitometric curve window. Phylogenetic analyses were performed using the Dice similarity coefficient (Dice, 1945) and the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on numbers and positions of bands by bionumerics (Sneath & Sokal, 1973). Gel-purified LpF1 was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) and sequenced using the M13 forward (−20) (5′-GTAAAACGACGGCCAG-3′), M13 reverse

(5′-CAGGAAACAGCTATGAC-3′), P1-FBA1 (5′-CAGATGGTCAATCAACGATC-3′), KU-60019 in vitro and P2-FBA1 (5′-CCGGGTGGTGGATTTAAACC-3′) primers using a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems) in a 3730 Genetic Analyzer (Applied Biosystems). LpF1 was subsequently characterized by sequence similarity searches against the GenBank database using the blast

algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997). The FBA1-specific fragment (LpF2) was amplified using 35 ng of template DNA, P3-FBA1 (5′-TCTATAATTTGTGATACAGGGGTTGCC-3′), and P4-FBA1 (5′-CTCGTAATCACACAGAAATTATGCTGC-3′) under the following cycling conditions: an initial 94 °C for 3 min; 35 cycles at 94 °C for 15 s, 59 °C for 35 s, and Z-VAD-FMK supplier 68 °C for 2 min; and a final 68 °C for 7 min. Genomic DNA (1 μg) from L. paraplantarum strains digested by Dra I were separated by a 1% agarose gel and transferred to nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). The LpF2 fragment (946 bp) was purified using a PCR purification kit (Qiagen) and labeled using a Digoxigenin (DIG) High Prime Kit (Roche Diagnostics GmbH) according to the manufacturers’ instructions. Hybridization was carried out at 42 °C. Membrane was washed under conditions of high stringency at 68 °C. Detection was

Metalloexopeptidase performed using an anti-DIG antibody alkaline phosphatase conjugate and CSPD. Membrane was activated at 37 °C for 10 min and developed to an X-ray film (Roche Diagnostics GmbH). Strains were preliminarily classified by sequence analyses of pheS, rpoA (Naser et al., 2005), and 16S rRNA genes (Table 1) and further confirmed using PCR-based methods (Berthier & Ehrlich, 1999; Torriani et al., 2001a, b). To discriminate these strains, we evaluated repetitive element sequence-based (REP-) (Jersek et al., 1999), triplicate arbitrarily primed (TAP-) (Cusick & O’Sullivan, 2000), RAPD-, and ERIC-PCRs, but those except ERIC did not yield a band that was specific to L. paraplantarum strains (data not shown). In ERIC-PCR, the L. paraplantarum strains tested had similar band profiles (Fig. 1a, lanes 7–13); the shared bands agreed with the type strain of L. paraplantarum (JCM 12533T, lane 7). The DNA bands of approximately 2.8, 1.1, 0.9, and 0.55 kb generated with the primer set ERIC-1R and ERIC-2 were common to strains of the species L. paraplantarum (Fig. 1a, horizontal arrows).

The study was approved by the University of East Anglia Ethics Committee. An introductory e-mail was sent out to 10,000 e-mail addresses held by the Centre for Pharmacy Postgraduate Education

containing a link to an online survey with a follow up e-mail after two weeks. It was estimated that 1/3rd of e-mail addresses may be no longer active and that only 50 % of the remaining e-mail addresses were for practicing community pharmacists. Participants were asked to enter how many consultations (one to one discussions in the consultation room) they had held with patients in their last standard working week. STATA® 12 SE was BGB324 ic50 used to conduct a backward stepwise elimination linear regression model for the number of consultations as the dependent variable. A total of 700 responses (42% of predicted potential www.selleckchem.com/products/ch5424802.html respondents) with 595 responses eligible for inclusion.

Descriptive results have been reported previously2. The median (quartiles) for the number consultations performed in a standard week was 5 (3, 10), these include Medicine Use Reviews, New Medicine Service and additional enhanced services such as emergency contraception. The statistically significant predictors of number of consultations in the final model were: working in a multiple pharmacy, having received consultation skills training during preregistration, male gender,

requesting further consultation skills training, and greater confidence in consultation skills. Confidence in consultations skills had the highest positive relationship with number of consultations. Participants had to rate their how confident they were in their consultation skills on a scale where 1 was not confident and 5 was fully confident. A value of 3 on the confidence scale was modelled 4-Aminobutyrate aminotransferase as having an increase of 34% in the number of consultations compared to the reference group of confidence 1 or 2 (p = 0.025); a value of 4 an increase of 56% (p < 0.001) and a confidence rating of 5 an 81% increase on the reference group (p < 0.001). The model explained 27.2% of the variance in the number of consultations. This exploratory analysis suggests that the more confident a participant is in their consultation skills, the more consultations they conduct. Previous research suggests that training is important in increasing confidence3. While there are many changes in pharmacy education to include consultation skills training during undergraduate and pre-registration year, there are still a large number of registered pharmacists for whom further training in consultation skills could help increase the delivery of more patient facing services.

The PhoU mutant identified in our previous transposon mutant screen has the transposon inserted near the C terminus of the phoU gene and has a more obvious persister phenotype than the phoU deletion mutant (Y. Li & Y. Zhang, unpublished data). Thus the finding that the PhoU deletion mutant selleck compound did not come up in our screen may be due to compensatory changes or mutations, which may indicate a limitation of the deletion mutant library approach. Like the PhoU mutant (Li & Zhang, 2007), the sucB and ubiF mutants have increased susceptibility to various stresses and different antibiotics with a two- to fourfold decrease in MIC and MBC (Table 1). It is generally assumed that mutations in genes involved

in persistence should not affect the MIC (Hansen et al., 2008). However, this may not necessarily be true. It is possible that mutation in a persister Lumacaftor molecular weight gene can affect antibiotic susceptibility not only in persisters but also in growing bacteria. As the current MIC and MBC testing is performed with a standard inoculum of 105–106 organisms of log phase cultures that may contain some persister bacteria already, it is likely that persisters may contribute to the MIC and MBC under normal MIC/MBC testing conditions. When the standard inoculum is inoculated into the culture medium containing antibiotics for MIC/MBC

testing, the mutants with defective persister formation are killed more rapidly than the wild-type bacteria at a given antibiotic concentration in the medium and therefore have lower MIC/MBC. In fact, all our persister-defective mutants, including phoU identified in the previous study (Li & Zhang, 2007) and ubiF and sucB identified in this study, have about two- to fourfold lower MIC/MBC than Coproporphyrinogen III oxidase the wild-type strain. A recent study, using the E. coli Keio mutant library screen to identify persistence genes with a short ofloxacin exposure of 6 h, found primarily stress response genes dnaJ and

dnaK (chaperones), apaH (diadenosine tetraphosphatase involved in stress resistance), surA (peptidyl-prolyl cis–trans isomerase involved in stationary phase survival), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (RNA polymerase-binding transcription factor, a positive regulator of RpoS), ygfA (5-formyl-tetrahydrofolate cyclo-ligase) and yigB (FMN phosphatase) (Hansen et al., 2008). As we indicated previously (Li & Zhang, 2007), persisters are highly variable and have to be defined by specific conditions. Persisters may consist of different subpopulations of varying hierarchy in continuum (Zhang, 2007), and different times of antibiotic exposure may lead to different persister populations, with longer exposure causing increasingly fewer persisters, which can be called ‘deep persisters’ (with lower metabolism), which are not killed by antibiotics even with long antibiotic exposure.

Both parasitological diagnosis and follow-up assessments of visceral leishmaniasis were based on molecular methods; i.e. PCR on peripheral blood (PB) [4] and less frequently on bone marrow (BM). Biological diagnosis was also based on PB and BM culture Selleckchem Verteporfin on blood agar/Novy–McNeal–Nicolle medium and direct microscopic examination of BM. Biological follow-up also included CD4 cell counts and HIV viral load measurements. Clinical follow-up of patients with visceral leishmaniasis and definitions of subclinical or clinical visceral leishmaniasis episodes have been described previously [4]. Additional quantitative real-time PCR tests were performed using a LightCycler™ instrument

with SYBRGreen (Roche, Meylan, France) for detection. All acquired fluorescence data were analysed using the LightCycler™ software. Melting curve analysis was used for characterization of the quantitative real-time PCR products. The primers, previously described in Mary et al. [7], amplified a kinetoplastic-specific sequence of 137 bp. Among the 27 Leishmania/HIV-coinfected patients followed up, 16 patients presented relapses and 11 were free of relapses. No clinical relapse occurred when CD4 cell counts were >200 cells/μL. Moreover, PCR analysis confirmed that the PB of nonrelapsing patients became Obeticholic Acid cell line definitively PCR-negative

in the first 6 months of follow-up [4]. As regards relapsing patients, 10 of them presented a total Rho of 52 relapsing visceral leishmaniasis clinical episodes, despite adequate drug treatment of both visceral leishmaniasis and HIV-1 infections. It is noteworthy that visceral leishmaniasis relapses are responsible for serious difficulties in the monitoring of coinfected patients [3–5]. Figure 1 shows the clinical evolution of seven of these 10 patients, indicating clinically relevant and subclinical episodes or periods without any signs of visceral leishmaniasis. Anti-leishmanial treatment and HAART, CD4 cell counts, occurrences of other opportunistic infections, and Leishmania PCR and culture results are also

shown in Figure 1. The median period of follow-up was 87.5 months (ranging from 5 to 158 months). During the follow-up period, seven patients died, one was lost to follow-up and two survived. All patients experiencing visceral leishmaniasis episodes received induction treatment with amphotericin B, miltefosine or pentamidine. For all patients, during each visceral leishmaniasis clinical episode, the PCR assay used for routine diagnosis detected circulating parasites (n=153), and most CD4 counts were <200 cells/μL. Acute episodes were followed by relapse-free periods with subclinical signs or without any symptoms of visceral leishmaniasis. During these periods, the patients were not given induction treatment, but primarily received secondary prophylaxis with amphotericin B or miltefosine (Fig. 1).

The spores of A. niger were counted in a Neubauer chamber and cells of the B. cepacia were quantified using a spectrophotometer (600 nm), using a previously established growth curve. Serial dilutions of the suspensions were made to obtain 22.3 × 106 spores mL−1 and 4.6 × 109 bacteria mL−1, and these were used as inoculum for the growth assays. The inocula concentrations were based on previous works (Barroso & Nahas, 2006; Park et al., 2010). The spore suspension (0.5 mL) and the cell suspension (0.75 mL) were inoculated into 50 mL sterile liquid media in Petri dishes (150 mm diameter) and incubated at 30 °C for 9 days without agitation. Both spore and cell suspensions were inoculated into co-cultures. Cultures were harvested

at 3-day http://www.selleckchem.com/products/ganetespib-sta-9090.html intervals by filtration or centrifugation. The mycelia were filtrated using Whatman No. 1 filter paper, preweighed, and dried; the filtrate was used for subsequent chemical analysis. The mycelium retained on the filter

paper was washed AG-014699 mouse with 50 mL of 1 M HCl to remove any undissolved CaP, followed by 50 mL of water and subsequently weighed; after which, the mycelia were dried in an oven at 105 °C for 24 h. The B. cepacia culture was centrifuged for 15 min at 10 000 r.p.m., and the supernatant was removed for chemical analysis. After removing the supernatant, the cells were washed with 10 mL of HCl 1 M. The pellet was resuspended in 10 mL of water; the dry weight of cell was assayed after drying at 105 °C for 24 h. The dry weight of a co-culture of A. niger–B. cepacia was obtained after centrifugation, using the same protocol described previously for the bacterial culture. Each treatment was replicated three times. The cell-free filtrates were used for chemical analysis. Soluble phosphate was determined using the Ames

(1966) method and quantified spectrophotometrically at 820 nm based on a standard curve. Titratable acidity and pH values were determined by titration of 10 mL of culture Branched chain aminotransferase medium with 0.02 M NaOH using an automatic Titration Manager. Residual sugar was determined spectrophotometrically at 607 nm using anthrone solution. The efficiency of solubilization (ES) of CaPO4 (expressed as a percentage) was determined by the relationship between the amount of phosphate solubilized and the amount added to the culture medium. Acid phosphatase activity was determined using 4 mM p-nitrophenylphosphate in 0.1 M acetate buffer (pH 5.4) as substrate (Nahas et al., 1982). The reaction mixture contained 0.2 mL of culture medium and 1.8 mL of substrate solution. The mixture was stirred and incubated for 20 min at 37 °C. The p-nitrophenol released was measured using a spectrophotometer at 405 nm. One unit of phosphatase activity was defined as the amount of enzyme required to liberate 1 μmol p-nitrophenol per hour under the assay conditions. Specific activity was expressed as units per mg dry biomass. All chemical analysis was performed in duplicate.

, 2006). Plant pathogenic oomycetes appear to have evolved a protein translocation system similar to malaria, which involves secreted proteins possessing an RxLR motif located after the signal peptide sequence (Bhattacharjee et al., 2006; Birch et al., 2006; Haldar et al., 2006; Whisson et al., 2007; Dou et al., 2008b). It was found that the RxLR motif is required for translocating these proteins into the host cells of infected plants (Whisson et al., 2007; Dou et al., 2008a). Bioinformatic analysis has shown that over 500 putative RxLR effectors are found in the potato pathogenic oomycete Phytophthora

infestans, and similarly, hundreds more in other plant pathogenic oomycetes (Whisson et al., 2007; Haas et al., 2009; Tyler, 2009). Sotrastaurin order It was demonstrated that the oomycete RxLR motif is functional in Plasmodium, where it can direct an RxLR–GFP fusion protein from the parasite into the host erythrocyte (Bhattacharjee et al., 2006). The PEXEL motif is also functional in P. infestans as it is able to translocate an avirulent chimaeric PEXEL-PiAvr3 protein into PiAvr3-recognizing potato plants (Grouffaud et al., 2008). Replacement of the N-terminal region of the effector protein PsAvr1b with a PEXEL motif HSP inhibitor containing leader sequences of three Plasmodium effectors resulted in the translocation of chimaeric

PsAvr1b into the Rolziracetam soybean cytoplasm (Dou et al., 2008a). Before detailed molecular interaction studies between Saprolegnia and fish can be performed, it is essential to develop a suitable infection model. The ami-momi treatment

established, which involves shaking fish in a net for approximately 2 min to remove part of the mucosal layer and subsequently challenging with Saprolegnia zoospores (Hatai & Hoshiai, 1993), is a good method to characterize the virulence of S. parasitica strains (Stueland et al., 2005). However, it is not a suitable model to study the early cellular and molecular infection mechanisms and events. In order to study these in more detail, the development of a fish cell-line infection assay is necessary. Here, we describe the identification and molecular characterization of a putative effector protein, SpHtp1, containing an RxLR motif. Microscopic studies of a Saprolegnia–fish interaction using an in vitro system involving a rainbow trout cell line showed that SpHtp1 is translocated into the fish cells, also when applied exogenously. A Saprolegnia parasitica isolate CBS223.65, obtained from the Centraal Bureau voor Schimmelcultures (CBS), the Netherlands, was grown on potato dextrose agar (Fluka) for 5 days at 18 °C, before inoculation in pea broth (125 g L−1 frozen peas, autoclaved, filtered through cheese cloth, volume adjusted to 1 L and autoclaved again) and incubation for 2 days at 24 °C. To accomplish S.

Quantitative invasion assay values were calculated as follows: We used an in vitro assay according to Trombert et al. (2010), modified from the method described by McCormick et al. (1993). Briefly, the colon carcinoma HT-29 cell line was grown to confluence (18–21 days) on 3.0-μm Cobimetinib datasheet pore-size filters (or transwells, Millicell®, Millipore) with glucose-free RPMI (Gibco). Each transwell was inoculated individually to the

apical surface with 400 μL of approximately 1 × 107 CFU mL−1 of bacterial cultures and immediately incubated for 60 min at 37 °C. After extensive washing with sterile PBS, the extracellular bacteria were killed by treatment of monolayers with gentamicin (50 μg mL−1). Immediately after gentamicin treatment, the medium from basal compartment

of the epithelial cell monolayer was collected and plated for CFU to assess the number of bacteria that passed through the cell monolayer. The polarization of cells was confirmed by transepithelial electrical 5-FU price resistance and transmission electron microscopy (data not shown). All results are expressed as means±SD of an individual experiment performed in triplicate. P-values were calculated according to Student’s t-test, and P<0.05 or P<0.01 values were considered statistically significant. To assess whether the sopD2 locus is a pseudogene in the serovar Typhi, we compared the available sequences of S. Typhi CT18 and S. Typhi Ty2 (McClelland et al., 2001; Parkhill et al., 2001; Deng et al., 2003). We observed that the sequence of sopD2 in S. Typhi contains a frameshift at codon 48 resulting in

a premature stop codon that disrupts the expected ORF. Accordingly, the online databases report sopD2 as a pseudogene (Fig. 1). To confirm the presence of this frameshift in our S. Typhi clinical strain collection, PCR assays were carried out using SopD21 and SopD22 primer pairs. The primers yield an 1800-bp amplicon in both S. Typhi and S. Typhimurium and were used to test the clinical strains obtained from Chilean typhoid patients (STH collection, Hospital Dr Lucio Córdova, Chile). The PCR products were sequenced and in silico comparison was performed with the reference strains (S. Typhi CT18, S. Typhimurium LT2 and Salmonella enterica serovar Paratyphi A ATCC 9150) using Farnesyltransferase the bioinformatic available program (clustalw; http://www.ebi.ac.uk/Tools/msa/clustalw2/). Our results indicated that the deletion described in S. Typhi CT18 is conserved among S. Typhi clinical strains (Fig. 2). Therefore, the sopD2 frameshift mutation seems to be a feature in the genome of serovar Typhi. Recently, we reported that S. Typhi harboring the sseJ gene from S. Typhimurium significantly disrupted HT-29 monolayer compared with the wild-type strain (Trombert et al., 2010). In the same way, we infected polarized HT-29 monolayers with the S. Typhi wild-type and S. Typhi sopD2STM strains.