Cell 1997,91(3):347–356 PubMedCrossRef 43 Morimatsu K, Kowalczyk

Cell 1997,91(3):347–356.PubMedCrossRef 43. Morimatsu K, Kowalczykowski SC: RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Mol Cell 2003,11(5):1337–1347.PubMedCrossRef 44. Lusetti SL, Cox MM: The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu Rev Biochem

2002, 71:71–100.PubMedCrossRef 45. Levine MM, Tacket CO, Sztein MB: Host- Salmonella interaction: human trials. Microbes Infect 2001,3(14–15):1271–1279.PubMedCrossRef 46. Tacket CO, Hone DM, Curtiss R III, Kelly SM, Losonsky G, Guers L, Harris AM, Edelman R, Levine MM: Comparison of the safety and immunogenicity of Δ aroC Δ aroD and Δ cya Δ crp Salmonella Typhi Necrostatin-1 strains in adult volunteers. Infect Immun 1992,60(2):536–541.PubMed 47. Frey SE, Bollen W, Sizemore D, Campbell M, Curtiss R III: Bacteremia associated with live attenuated χ8110 Salmonella VX-680 supplier enterica serovar Typhi ISP1820 in healthy adult volunteers.

Clin Immunol 2001,101(1):32–37.PubMedCrossRef 48. McClelland M, Sanderson KE, Clifton SW, Latreille P, Porwollik S, Sabo A, Meyer R, Bieri T, Ozersky P, McLellan M, et al.: Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid. Nat Genet 2004,36(12):1268–1274.PubMedCrossRef 49. Deng W, Liou SR, Plunkett G III, Mayhew GF, Rose DJ, Burland V, Kodoyianni V, Schwartz DC, Blattner FR: Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J Bacteriol 2003,185(7):2330–2337.PubMedCrossRef 50. Espinosa-Aguirre J, Barajas-Lemus C, Hernandez-Ojeda S, Govezensky T, Rubio J, Camacho-Carranza R: RecBCD and RecFOR dependent induction of chromosomal deletions by sodium selenite in Salmonella . Mutat Res 2009,665(1–2):14–19.PubMed 51. Cano DA, Pucciarelli MG, Garcia-del

Portillo F, Casadesus J: Role of the RecBCD recombination pathway in Salmonella virulence. J Bacteriol 2002,184(2):592–595.PubMedCrossRef 52. Buchmeier NA, Lipps CJ, So MY, Heffron F: Recombination-deficient mutants of Salmonella Typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol Microbiol 1993,7(6):933–936.PubMedCrossRef 53. Bertani G: Studies on Florfenicol lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli . J Bacteriol 1951,62(3):293–300.PubMed 54. Sun W, Wang S, Curtiss R III: Highly efficient method for introducing successive multiple scarless gene deletions and markerless gene insertions into the Yersinia pestis chromosome. Appl Environ Microbiol 2008,74(13):4241–4245.PubMedCrossRef 55. Roland K, Curtiss R III, Sizemore D: Construction and evaluation of a Δ cya Δ crp Salmonella Typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis 1999,43(3):429–441.PubMedCrossRef 56.

5 spectra [19] The plot in Figure  1b illustrates the max(J tot)

It should be noted that although only b/Λ is given in the figure, the results are actually from a number of 2D parametrical sweep for both Λ (from 300 to 1,100 nm with step 50 nm) and b/Λ (from 0.5 to 1 with step 0.05), i.e., the 3D PV system has been simulated for hundreds of times in order to find the designs with the highest J tot. For

Selleck MGCD0103 each b/Λ, only the maximized J tot under an optimized Λ, which generally varies under different b/Λ, is recorded. Compared to the planar cell (i.e., b/Λ = 1) with J tot approximately 20.79 mA/cm2, two-dimensionally nanopatterning top junction always leads to a much higher J tot with a peak of 27.69 mA/cm2 (see red curve

for unpolarized case) at b/Λ = 0.75, Selleck LY2109761 Λ x  = 450 nm, and Λ y  = 850 nm. In addition, transverse electric (TE, i.e., electrical field E along y) and transverse magnetic (TM, i.e., E along x) incidences show identical max(J tot) due to the geometrical symmetry, while the value for unpolarized, i.e., (TE + TM)/2, is generally lower. To explore the physics behind the above observation, contour maps of max(J tot) versus Λ x and Λ y are given in Figure  2a,c for TM, TE, and unpolarized cases, respectively. In these figures, b/Λ = 0.75 is used according to the design of Figure  1 and the peaked J tot values in mA/cm2 have been marked directly. Comparing Figure  2 panels a and b, the photocurrent maps for TE and TM cases are mutually symmetrical with respect to the line of Λ y  = Λ x . This is rational since it is completely equivalent to rotate either the electric polarization or the device by 90° in the x-y plane. This answers the question that why the curves (in blue) for TE and TM are undistinguishable in Figure  1b. However, J tot is not peaked under the same

grating pitches for TE or TM (see Figure  2a,b). A direct Branched chain aminotransferase consequence is that the maximal J tot for unpolarized illumination cannot reach the value under linear polarization. This can be seen from Figure  2c, where max(J tot) = 27.72 mA/cm2 (<28.05 mA/cm2 from linear case) is found at Λ x  = 520 nm and Λ y  = 930 nm. It should be noted that the peaked value and optimal pitches are slightly changed from Figure  1b since a finer sweep with Λ step of 10 nm is employed. Figure 2 Grating pitch optimization and absorption spectra. J tot versus Λ x and Λ y for (a) TM, (b) TE, and (c) (TE + TM)/2; (d) J tot versus Λ y at Λ x  = 520 nm with planar case (wo, i.e., without nanopattern design) for reference; P abs versus Λ y and λ under (e) TM and (f) TE incidences, where Λ x  = 520 nm. b/Λ = 0.75 (according to Figure 1) is used in all figures. Figure  2d plots J tot as a function of Λ y with b/Λ = 0.75 and Λ x  = 520 nm for all interested polarizations conditions. Also inserted is the J tot of the planar system mentioned previously.

Figure 5 Generation of tumor-specific CTLs ex vivo Splenic CD3+

Figure 5 Generation of tumor-specific CTLs ex vivo. Splenic CD3+ T cells were isolated from B6 mice with MACS. T cells were primed with MAGE-1-modified DCs as described in Materials and Methods. DC-Ad-LacZ and untreated DCs were used as controls. Primed T cells (effector cells) were titrated by serial dilution, then mixed with MFC or B16F10 target cells, and their lytic activity was assayed. Results are given as means ± SD from three independent experiments. A therapeutic effect mediated by DC-Ad-MAGE-1 in vivo Therapeutic

potential of DC-Ad-MAGE-1 was further explored with an JNK-IN-8 purchase established tumor model. 5 × 105 MFC or B16F10 tumor cells were implanted s.c in B6 mice, and tumor-bearing mice were injected with different modified or unmodified DCs on days 5 and 12. Fig. 6A shows that tumor growth was significantly inhibited in mice vaccinated with DC-Ad-MAGE-1. For example, tumor volumes on day 27 were as follows: untreated DC control 14.98 ± 1.81 cm3, DC-Ad-LacZ control 15.44 ± 1.99 cm3, DC-MFC Ag control 7.79 ± 1.55 cm3, DC-Ad-MAGE-1 3.46 ± 1.12 cm3, DC-Ad-MAGE-1 vs. the other control groups (P < 0.05). Half of the tumor-bearing mice immunized with DC-Ad-MAGE-1 survived in a period of over 60 days. By contrast, only AC220 order 10% of the tumor-bearing

mice immunized with DC-MFC Ag survived; all mice from the other control groups succumbed to growing tumors within 25 days, thus providing no therapeutic effect (Fig. 6B). The differences between the DC-Ad-MAGE-1 group and all control groups were statistically significant (P < 0.05).

Figure 6 Inhibition of tumor growth in tumor-bearing mice by immunization with MAGE-1-modified, CCL3 and CCL20-recruited DC vaccine. (A), Each of 10 mice in a group was challenged s.c. with 1 × 105 viable MFC tumor cells. Mice were subsequently injected s.c. with DC-Ad-MAGE-1 5 filipin days later. As controls, tumor-bearing mice were injected with DC-Ad-LacZ, DC-MFC Ag, or untreated DCs. Survival was observed over time after immunization of mice harboring preexisting tumors. Survival rate was compared with a long-rank test of Kaplan-Meier curves. (B), Tumor growth was measured every 2~3 days after the second immunization. Data are given as means ± SD of 10 mice per group from three independent experiments. To confirm that tumor-specific CTLs had indeed been generated in the immunized mice, the following evaluation was performed. Spleen T cells from mice immunized s.c with DC-Ad-MAGE-1, and thus rendered tumor-free after MFC tumor challenge, were restimulated ex vivo with irradiated tumor cells and tested for cytolytic activity. As shown in Fig. 7A, these effector cells efficiently lysed MFC, but not B16F10 tumor cells. Control spleen T cells from naive mice stimulated with irradiated MFC tumor cells failed to demonstrate CTL activity. Furthermore, splenic CD3+ T cells derived from those mice that survived MFC challenge produced high levels of IFN-γ, but not when stimulated with B16F10 cells (Fig. 7B).

Multiplex PCR performed using ompA, csuE, and bla OXA-51-like as

Multiplex PCR performed using ompA, csuE, and bla OXA-51-like as target genes [24] confirmed these differences (data not shown). Biofilm formation by A. baumannii clinical isolates The A. baumannii isolates belonging to the SMAL

clone were tested for NU7026 price their ability to form biofilm, measured as surface adhesion to polystyrene microtiter plates. Biofilm growth is considered an important factor for host colonization [25, 26] and for resistance to environmental and cellular stresses [11]. Ability to form biofilm, measured as surface adhesion to polystyrene microtiter plates, was very similar for all A. baumannii isolates tested (data not shown); results shown throughout the paper refer to the A. baumannii isolate described in Line 22 of Table 1. This isolate selleck chemicals was considered

representative of the A. baumannii SMAL clone since it belongs to the main genotypic subgroup of the SMAL clone (Figure 1) and since it was the first A. baumannii to be isolated in this survey. Surface adhesion to microtiter plates by A. baumannii SMAL clone was determined in various growth conditions, comparing two growth temperatures (30°C vs. 37°C), and different growth media: the rich peptone-based LB medium, LB medium diluted 1:4 (LB1/4), the M9Glu/sup medium [[27], described in Methods], and the M9Suc/sup in which 0.2% sucrose was added as main carbon source instead of glucose. LB1/4 was tested since it was shown to promote production of adhesion factors in other Gram negative bacteria, such as Escherichia coli [28]. We found that biofilm formation by A. baumannii SMAL was strongly affected both by growth media and by temperature: indeed, while surface adhesion was very poor in LB medium at either 30°C or 37°C, it was clearly stimulated by growth in LB1/4, although only

at 30°C. Finally, growth in M9Glu/sup resulted in efficient surface adhesion both at 30°C and at 37°C, while growth selleck compound in sucrose-based medium (M9Suc/sup) resulted in much lower levels (Figure 2A). The observation that growth temperature affects biofilm formation in the LB1/4, but not in sugar-based media such as M9Glu/sup, would suggest that this process could be mediated by different mechanisms and by different adhesion factors. Figure 2 A. Surface adhesion to polystyrene microtiter plates by A. baumannii SMAL clone. Black bars bacterial cultures grown in LB medium; light grey bars LB1/4 medium; white bars M9Glu/sup; dark grey bars M9Suc/sup. B. Binding of Calcofluor to A. baumannii SMAL clone grown in solid media. C. Inhibition of A. baumannii biofilm formation by cellulase treatment: circles, M9Glu/sup medium; diamonds, M9Suc/sup medium; squares, LB1/4 medium. The horizontal dotted line indicates the 50% inhibition mark. IC50′s values are indicated by vertical dotted lines. A major adhesion factor characterized in A. baumannii is represented by the csu pili described in the A. baumannii strain ATCC 19606 [17].

Nevertheless, as Steinert and Snell [3] indicate interactive appr

Nevertheless, as Steinert and Snell [3] indicate interactive approaches require utilization of various forms of questioning which “”can Fosbretabulin cell line stimulate interest, arouse attention, serve as an ‘ice-breaker’ and provide

valuable feedback to the teacher and student alike”". Questioning and probing students effectively are skills that educators should be trained on during teaching enhancement programs for Faculty [22, 23]. The dynamics of the tutorial process is multifaceted including the educational methods, the tutor, and the learners. Concentrating on one of them will lead to an incomplete understanding of the educational process [24]. Thus, it is important to take a holistic approach to evaluate teaching and learning. This opinion was supported by others [25]. Contemporary instructional strategies that considers only instructor behaviors, is unlikely to succeed in improving the quality of education. Action

should be done at the same time on educational methods and promoting LGX818 active students’ learning. We tried to achieve that by developing an educational tool which actively involves the students in the learning process. In summary The interactive problem-solving approach for tutorials can be an effective enjoyable alternative or supplement to traditional instruction for teaching traumatology to medical students. Training for this approach should be encouraged for Faculty development. Consent An informed consent was taken from patients to use their images for medical

education/publication. References 1. Goldstein GS, Benassi VA: Students’ and instructors’ beliefs about excellent lecturers and discussion leaders. Research in Higher Education 2006, 47:685–707.CrossRef 2. Brown G, Manouge M: AMEE Medical Education Gudie No 22: refreshing lecturing: Megestrol Acetate a guide for lecturers. Med Teach 2001, 23:231–234.CrossRefPubMed 3. Steinert Y, Snell LS: Interactive lecturing: strategies for increasing participation in large group presentations. Med Teach 1999, 21:37–42.CrossRef 4. Norman GR, Schmidt HG: The psychological basis of problem-based learning: a review of the evidence. Acad Med 1992, 67:557–565.CrossRefPubMed 5. Marsh HW: Students’ evaluations of university teaching: Research findings, methodological issues and directions for future research. Int J Educ Res 1987, 11:255–388.CrossRef 6. Johns M: Design of slides. J Audiov Media Med 1995, 18:121–128.PubMed 7. Cox KR, Ewan CE: Designing illustrations for teaching. In The Medical Teacher. Edited by: Cox KR, Ewan CE. Edinburgh, Churchill Livingstone; 1982:144–149. 8. Centre for Professional Development: S.E.C.A.T Student evaluations of courses and teaching booklet. The University of Auckland, Auckalnd, New Zealand; 1996:8–11. 9.

Because of this, disadvantages appear in realizing an efficient S

Because of this, disadvantages appear in realizing an efficient Si NC light-emitting diode (LED). To realize efficient Si NC LEDs, therefore, following required factors such as the formation of Si NCs with high density, surrounding matrix, and design of an efficient carrier injection film

should be Seliciclib mouse addressed. We and others have recently demonstrated an in situ growth of well-organized Si NCs in a Si nitride (SiN x ) matrix by conventional plasma-enhanced chemical vapor deposition (PECVD) and have achieved a reliable and stable tuning of the wavelength ranging from near infrared to ultraviolet by changing the size of Si NCs [8, 10, 11]. SiN x as a surrounding matrix for Si NCs can provide advantages over generally used Si oxide films because of the in situ formation of Si NCs at low temperature, small bandgap, and clear quantum confinement dependence on the size of Si NCs. These merits can meet the requirements Angiogenesis inhibitor for the current CMOS technology such as compatibility with integration and cost-effectiveness. To inject the carriers into the Si NCs, polysilicon, indium tin oxide (ITO), and semitransparent metal films have been generally used as contact materials [12–14]. However, the photons generated from the Si NCs could be absorbed because the photons passed through these contact materials

to escape out from the Si NC LEDs. A suitable carrier injection layer is, therefore, very crucial for enhancing the light emission efficiency of Si NC LEDs. In previous results [15, 16], we grew the amorphous SiC(N) film with an electron density up to 1019 cm−3 using a PECVD at 300°C and demonstrated that the amorphous SiC(N) film could be a suitable electron injection layer to improve the light emission Niclosamide efficiency of Si NC LEDs. Recently, alternative methods such as surface plasmons (SPs) by nanoporous Au film [17] or Ag particles [18] that could enhance the luminescence efficiency from the Si NCs and external quantum efficiency of a Si quantum dot LED were reported. These approaches, however, need complicated wet etching and annealing processes

to apply SP coupling. They also have disadvantages in realizing an efficient Si NC LED, such as having an impractical structure for LED fabrication and absorption of light escaping out from the LED at the metal layer. A reliable, simple, and practical device design without additional processes is, hence, very crucial in the fabrication and an enhancement of the light emission efficiency of Si NC LED. In this work, we present the concept that can uniformly transport the electrons into the Si NCs by employing 5.5 periods of SiCN/SiC superlattices (SLs) specially designed for an efficient electron transport layer, leading to an enhancement in the light emission efficiency of Si NC LED. A SiCN film in 5.5 periods of SiCN/SiC SLs was designed to have a higher optical bandgap than that of SiC to induce a two-dimensional electron gas (2-DEG), i.e.

Most topologies assigned all strains to the same main clades as i

Most topologies assigned all strains to the same main clades as in the whole genome phylogeny, with a few exceptions: 33-rpoB assigned F. hispaniensis to clade 2 and 19-iglC assigned W. persica to clade 2, in subgroup F. noatunensis subsp. orientalis (in both assignments). This is

an interesting observation as rpoB was recently suggested as an alternative marker to 16S rDNA in metagenomic studies [21]. The level of incompatibility and difference in resolution compared to the whole-genome reference topology were decreased, in some cases by a considerable amount, by selecting an optimal combination of markers. Moreover, topologies based on an optimal set of markers significantly increased the average HDAC inhibitor mechanism statistical support (i.e. average bootstrap). Generally, both the degree of compatibility and resolution were improved by concatenating sets of two to seven markers in all possible combinations. However, C188-9 ic50 some combinations, in particular

considering incompatibility, might result in poorer topologies than for an estimated topology based on a single marker. This observation is consistent with previous work where concatenation of sequence data have resulted in biased phylogenetic estimates [50]. All incompatible phylogenetic signals were removed in topologies based on optimised sets of two to seven markers, in contrast to random concatenation. Totally congruent topologies were obtained by concatenating as few as only two markers (08-fabH and 35-tpiA). These two markers were included in all optimal sets. Hence, by selecting an optimal set of markers, a large improvement in resolution and compatibility can be obtained over random concatenation. An exhaustive search strategy was employed to find the optimal set of markers since the total number of available markers was relatively small. It should be pointed out that the number of possible marker combinations increases rapidly with the number

of markers considered Urocanase and soon becomes computationally intractable. As all the 742 gene fragments of the core genome in the analysed population have recently become available in [3], an interesting extension to the current work would be to find the optimal set of markers based on all those genes. Such an optimisation could be carried out by utilising one of the myriad of available optimisation techniques, such as a simulated annealing approach [51, 52]. It should be noted that we do only try to minimize the value of the objective metrics, incongruence or resolution difference, with respect to the whole-genome topology. There is no guarantee that the whole genome topology accurately resembles the true underlying species topology as systematic errors and statistical inconsistencies in the phylogenetic inference method could be amplified when analyzing whole genome data [50, 53–55].

Methods Leishmania

from VL Thai patients Samples used in

Methods Leishmania

from VL Thai patients Samples used in this study were collected from five autochthonous VL patients reported from Phang-nga, check details Trang, Songkla, and Stun provinces, southern Thailand. All patients presented with hepatosplenomegaly and pancytopenia. Amastigotes were identified under microscope from Giemsa-stained bone marrow smears in all cases. Two axenic cultures of promastigotes were obtained using bone marrow aspirates in Schneider’s medium supplemented with 20% FBS. Genotypic characterization was processed on three positive clinical samples (i.e., Giemsa-stained bone marrow smears and buffy coat) and two cultured promastigotes. The information of these samples is shown in Table 1. Table 1 The characteristics of five samples of autochthonous leishmaniasis used in this study Isolates Location Year of isolation Clinical presentation of leishmaniasis HIV

coinfection Source of DNA Sequence accession no. [reference] SSU-rRNA ITS1 hsp70 cyt b CU1 Songkhla 2011 VL# Yes Culture JX195633 JX195639 KC202883 JX195635 PCM1+ Phang-nga 2007 VL Yes Bone marrow smear JN885899 [8] EF200012 [7] not sequenced JX195636 PCM2§ Trang 2010 CL* and VL Yes Culture JQ280883 [8] JX195640 KC202880 JX195634 PCM4 Stun 2010 VL No Bone marrow smear JN087497 JX195637 KC202882 not sequenced PCM5 Trang 2011 CL and VL Yes Buffy coat not sequenced not sequenced KC202881 not sequenced +, this isolate was previously described in the study by Sukmee et al. [7]; §, this isolate JNK-IN-8 solubility dmso was previously described as Trang strain in the study by Bualert et al. [8]; #, visceral leishmaniasis; *, cutaneous leishmaniasis. Ethics statement The study was approved by the Ethics Committee of the Royal Thai Army Medical Department, Thailand. No information on the patients was presented Protein tyrosine phosphatase in this study. DNA preparation DNA was extracted from the Giemsa-stained smears of bone marrow using modified FTA extraction paper (Whatman, Bioscience, USA) following the protocol as previously described [18]. The Genomic DNA Mini Kit (Tissue) (Geneaid, USA) was used to extract the DNA from other three remaining samples. PCR amplification PCR assays were used to amplify a

fragment of four genetic loci using the previously described conditions, i.e., SSU-rRNA [19], ITS1 region [20], hsp70 [21], and cyt b [22]. The PCR products were subjected to electrophoresis on 1.5% agarose gels and stained with SYBR safe (Invitrogen, USA). Gels were photographed and documented on high-density printing paper using Uvisave gel documentation system I (Uvitech, UK). Cloning and sequencing PCR products amplified from the four loci were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA) according to the manufacturer instructions and then directly sequenced. For the PCR products that had insufficient amounts of DNA for direct sequencing, they were cloned in E. coli competent cells to produce a higher quantity of identical DNA.

Colicin expression Another group of genes upregulated in iron-def

Colicin expression Another group of genes upregulated in iron-deficient conditions were the genes encoding the Microcin V (cvaA

cvaB cvaC) and Colicin Ia, which were also upregulated in human serum and urine. Previous reports have shown the influence of bacterial intracellular iron levels on colicin expression, but the reason of such induction is still poorly understood [29–31]. Of note, transcription of immunity protein for both colicins was not upregulated in any of the conditions studied except for Colicin Ia in human serum. Expression of ORFs of unknown function in iron-deficient environments Two ORFs with unknown functions, shiF and ORF 123, were upregulated in iron-deficient BMS202 order conditions, with large fold changes in vivo and ex vivo. ORF 123 was the most strongly upregulated (> 100-fold) in the 3 test conditions, and was expressed 3 to 4 times more strongly than the iron acquisition systems. A nucleotide homology search using the BLAST program [32]

showed that ORF 123 is highly homologous (99%) to an ORF present in E. coli plasmids possessing a CVP region (such pAPEC-O1-ColI-BM, pAPEC-O2-ColV and pAPEC-1) or located on the chromosome of UPEC strains such as CFT073 (ORF c1220; 94%) and 536 (ORF ECP–0281; 95%). No homologous gene is find more found in the commensal E. coli strain MG1655. Transcriptome analysis by Mobley et al.[16]

showed over-expression of c1220 transcripts in E. coli CFT073 in a mouse model of UTI. The putative protein encoded by ORF Lck 123 showed 45-50% identity to three phospho-2-dehydro-3-deoxyheptonate aldolases that catalyze the first reaction of the shikimate pathway and are present on the chromosome of E. coli K12. This pathway involves seven enzymatic reactions that generate chorismate, a factor involved in the synthesis of three aromatic amino acids (tyrosine, tryptophan and phenylalanine) [33]. However, this pathway is also involved in other reactions, such as biosynthesis of siderophore group nonribosomal peptides such as yersiniabactin and enterobactin. In plasmid pS88, as in other CVP-containing plasmids, ORF 123 lies just upstream of iroN and is preceded by a sequence resembling the Fur Box consensus sequence (5′-GATAATGATAATCATTATC) [34, 35]. BLAST analysis of complete genomes available on publicly available database showed that ORF 123 is only found when the salmochelin operon is present but the reciprocity is not true, as for example in strain UTI89, which harbors only an iro locus. On the chromosome of E. coli strains CFT073 and 536, this ORF (c1220 and ECP_0281, respectively) is located in a pathogenicity island containing an iro locus but is 20–30 kb distant from the iro locus.

8 to 1 6 nmol per mg of protein This corresponds to a decrease i

8 to 1.6 nmol per mg of protein. This corresponds to a decrease in intracellular concentration from 1.8 to 0.5 mM, assuming an intracellular volume of 3.2 mL/mg of protein, [8]). The drop (about 70%) was rapid, occurring in less than 30 min, but the subsequent decrease in ATP levels was slow, the intracellular concentration after several hours remaining ≥ 0.3 mM in spite of PI3K inhibitor the absence of a carbon source. This suggests that the bacteria are able to use endogenous energy sources (such as glycogen for instance) in order to maintain a minimal energy charge, allowing survival, but not growth. When AThTP was allowed to

accumulate for 4 h in the absence of a carbon source, addition of various metabolizable substrates induced a sharp decrease in AThTP content (inset

of Figure 1). As previously shown [2], glucose addition (10 mM) triggered a drop of 80-90% in AThTP in less than 5 min and nearly 100% after 30 min, while the decrease was slower with other carbon sources (especially succinate and acetate). We also confirmed that virtually no AThTP was produced when a metabolizable carbon source was present at zero time (when bacteria were transferred from LB to M9 medium). As shown in Table 1, glucose was very effective in antagonizing AThTP accumulation, as an external concentration as low as 1 mM reduced the AThTP content (measured after 60 min) by about 80% while a concentration ≥ 5 mM nearly completely prevented the accumulation of AThTP. However, at high ionic strength (1 M NaCl, EPZ5676 KCl or choline chloride), glucose was unable to prevent AThTP accumulation. This is not surprising, as the high ionic strength is known to impair glucose utilization by E. coli cells [9]. Table 1 Effect of various

carbon sources on AThTP production in the BL21 E. coli strain.   AThTP(pmol/mg of protein) Control 88 ± 6 D-Glucose (1 mM) 13 ± 4 D-glucose (2.5 mM) 9 ± 2 D-Glucose (5 mM) < 2 D-Glucose (10 mM) < 2 L-Lactate (10 mM) 14 ± 2 Succinate click here (10 mM) 6 ± 1 L-Malate (10 mM) 8 ± 2 D-Glucose (10 mM) + NaCl (1.2 M) 94 ± 13 D-Glucose (10 mM) + KCl (1.2 M) 92 ± 6 D-Glucose (10 mM) + Choline Cl (1.2 M) 131 ± 15 Streptomycina (10 μM) 62 ± 2 Neomycina (10 μM) 68 ± 3 AAb 12 ± 2 AAb + serine hydroxamate (0.5 mg/mL) 18 ± 2 aAll amino acids (40 μg/mL each) with the exception of serine bNo carbon source present The bacteria (A600 > 1) were incubated for 60 min at 37°C in minimal M9 medium containing substrates at the concentrations indicated. Mean ± SD for 3 – 9 experiments. The antibiotics streptomycin and neomycin have little effect on AThTP accumulation in the absence of a carbon source, suggesting that protein synthesis is not required for AThTP accumulation. We also wanted to know whether the appearance of AThTP was specifically linked to carbon starvation or could be triggered by other forms of nutritional downshifts or cellular stress.