(PPT 1 MB) References 1 Bourne HR, Sanders DA, McCormick F: The

(PPT 1 MB) References 1. Bourne HR, Sanders DA, McCormick F: The GTPase

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of Applied Microbiology 1997, 83:85–90.check details PubMedCrossRef 11. McLaughlin MR: Simple colorimetric GANT61 datasheet rnicroplate test of phage lysis in Salmonella enterica. Journal of Microbiological Methods 2007, 69:394–398.PubMedCrossRef 12. Fraser D, Crum J: Enhancement of mycoplasma virus plaque visibility by tetrazolium. [http://​aem.​asm.​org/​cgi/​reprint/​29/​2/​305]Applied Microbiology 1975, 29:305–306.PubMed 13. McLaughlin MR, Balaa MF: Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR). Journal of Microbiological Methods 2006, 65:318–323.PubMedCrossRef 14. Pattee PA: Use of tetrazolium for improved resolution of bacteriophage plaques. Journal of Bacteriology

1966, 92:787.PubMed 15. Hurst CJ, Blannon JC, Hardaway RL, Jackson WC: Differential effect of tetrazolium dyes upon bacteriophage plaque-assay titers. [http://​aem.​asm.​org/​cgi/​reprint/​60/​9/​3462?​view=​long-pmid=​16349397]Appl Environ Microbiol 1994,60(9):3462–3465.PubMed 16. Ackermann HW: 5500 Phages examined in the electron microscope. Archives of Virology 2007, 152:227–243.PubMedCrossRef 17. Somerson NL, Morton HE: Reduction of tetrazolium salts by pleuropneumonialike organisms. Journal of Bacteriology 1953, 65:245–251.PubMed 18. McLaughlin MR: Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Tacrolimus (FK506) Salmonella. Journal of Microbiological Methods 2006, 67:611–615.PubMedCrossRef 19. Krueger AP, Cohn T, Smith PN, Mcguire CD: Observations selleck compound on the effect of penicillin on the reaction between phage and staphylococci. Journal of General Physiology 1948, 31:477–488.PubMedCrossRef 20. Winston HP: Bacteriophage formation without bacterial growth: I. Formation of staphylococcus phage in the presence of bacteria inhibited by penicillin. The Journal of General Physiology 1947, 31:119–126.CrossRef 21. Winston HP: Bacteriophage formation without bacterial growth II. The effect of niacin and yeast extract on phage formation

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Photoluminescence spectra Figure 4 (a) shows the PL spectrum of Z

Photoluminescence spectra Figure 4 (a) shows the PL spectrum of ZnO films fabricated at 400°C using GaN buffer layer, and Figure 4 (b) shows the PL spectra of ZnO/Si thin film grown at 400°C.

Figure 4 shows three main emission peaks. One intense peak centered at 373 nm is near-band emission, which corresponds to the exciton emission from near conduction band to valence band. Another weak one located at 456 nm is defect emission. As shown in Figure 4, merely the weak defect emission band centered at 456 and 485 nm can be observed in two thin films. This blue emission located at 456 nm most likely derives from electronic transition from the donor level of Zn interstitial to acceptor energy level of Zn vacancy according to Sun’s calculation by full-potential linear see more muffin-tin orbital method [25–27]. This shows that some Zni atoms exist in fabricated ZnO thin films. The emission located at 485 nm may be caused by the electronic transition between the anti-oxygen (OZn) and the conduction band. The PL spectra in Figure 4 (a) show that the UV emission Metabolism inhibitor of ZnO thin film fabricated on GaN/Si substrate is higher than

that fabricated on the Si substrate. The ratio of intensity of UV emission of ZnO/GaN/Si film to that of ZnO/Si film is about 2:1, and the ratio of FWHM of UV peak of ZnO/GaN/Si film to that of ZnO/Si film is about 7:11. Figure 4 PL spectra of ZnO thin film deposited on different substrates at 400°C. (a) Si substrate and (b) GaN/Si substrate. As Liothyronine Sodium shown in Figure 4 (a), the UV emission located at 367 nm is increased, and the visible emission at 456 nm is decreased. The increase of UV emission and the decrease of the defect emission indicate that the structure of ZnO/GaN/Si thin film becomes more perfect. The UV peak appears as a redshift from 367 to 373 nm. The relaxation of interface strain is the main reason because of the formation of ZnO/GaN/Si heterostructure. The PL spectra of ZnO thin film fabricated on two different substrates show

that the PL MI-503 molecular weight property of thin film fabricated using GaN buffer layer is more superior to that of ZnO/Si film. The ratio of visible emission of ZnO thin film fabricated on Si substrate is high, indicating that more defects exists in ZnO thin film. This is consistent with the analysis of two XRD spectra of ZnO thin films above. Conclusion ZnO thin films have been fabricated on GaN/Si and Si (111) substrates at the deposited temperature of 400°C, respectively. The structural and optical properties of ZnO thin films fabricated on different substrates are investigated systematically by XRD, FESEM, FTIR, and PL spectra. The FESEM results show that the ZnO/GaN/Si film is two-dimensionally grown with flower-like structure, while the ZnO/Si film is the (002) orientation grown with an incline columnar structure. The GaN buffer layer plays an important role for the transformation of the growth mode of ZnO thin films from one-dimensional to two-dimensional.

An appropriate evolutionary adaptation of germinant receptor expr

An appropriate evolutionary adaptation of germinant receptor expression/regulation is thus crucial to allow the cyclic transition between sporulation and germination upon environmental changes. In the ABT-263 in vivo construction of the complementation mutants in our study, certain precautions were therefore taken to avoid extensive over-expression of the complemented germinant receptor genes. By including some of the flanking regions of the gerAA, gerAB and gerAC fragment in the complementation plasmid, we wanted to maintain the native regulatory elements

of this locus. In addition, a shuttle-see more vector with an expected low or moderate copy number was sought as a basis for the complementation plasmid. To our knowledge, there is no shuttle-vector available for B. licheniformis where the copy number is demonstrated to be low or moderate. However, Arantes and Lereclus

[52] have constructed the pHT315 E. coli/B. thuringiensis shuttle-vector, with a copy number of ~ 15 per equivalent B. thuringiensis chromosome. This vector Selleckchem Defactinib has successfully been used in germinant receptor complementation studies in B. megaterium [53], and was thus considered as a reasonable choice for B. licheniformis. Despite that this vector has shown to be stably maintained in B. thuringiensis and B. megaterium without a selective pressure [52, 54], the antibiotic erythromycin had to be included to ensure persistence of the complementation plasmid during sporulation of the B. licheniformis complementation mutant NVH-1311. This could be due to a different segregation stability of the vector in B. licheniformis. Another possibility is that there is a potential Sulfite dehydrogenase elevated risk of plasmid curing due to sporulation at a high temperature. Sporulation of B. licheniformis MW3, NVH-1307 and NVH-1311 were performed at 50 °C since a pilot study showed that sporulation at this temperature

was faster, yielded more stable spores (less spontaneous germination) and a higher percentage of phase bright spores (results not shown). Disruption of gerAA abolish L-alanine and casein hydrolysate induced germination Decrease in absorbance at ~ 600 nm (A600) is used as a convenient method to monitor and compare germination of different spore populations [55, 56]. A fall in absorbance reflects a change in the refractive index (light scattering) of the multiple individual spores in a suspension, associated with germination events such as the excretion of spore’s depot of Ca2+-DPA, followed by water influx, cortex degradation and core swelling [51, 56–59]. Figure 1 shows a representative experiment where different strains of heat activated (65 °C 20 min) spores (in Phosphate buffer) are supplemented with the germinant L-alanine. At these conditions, a clear change in absorbance was observed for spores of wild type (MW3) and wild type complementation mutant (NVH-1311) supplemented with L-alanine. Less than a 5%/h decrease in absorbance was observed for spores of the disruption mutant (NVH-1307).

Furthermore, in some of the experiments the promoter activity was

Furthermore, in some of the experiments the promoter activity was almost abolished for construct B, while other experiments showed only a low activity. The part of the promoter click here retained in construct A but lost in construct

B contains no known putative binding sites for transcriptional regulators. It should be noted that the differences of expression between the longer promoter fragments (constructs A-D) were significant within experiments (three independent measurements) but not always between the experiments. However, all experiments showed the same general expression pattern for fragments A-D even though the actual levels differed. The difference between the longer promoter fragments (construct learn more A-D) and the shortest fragment (construct E) were significant between all experiments. As expected, the positive control pPrbcL-gfp showed very high expression levels in all experiments (data not shown). Figure 4 Expression from the hupSL promoter deletions. Measurements of GFP fluorescence intensity

in living cells grown under nitrogen fixing conditions. Nostoc punctiforme ATCC 29133 cells were transformed with vector constructs containing truncated versions of the hupSL-promoter (A-E) fused to the reporter gene gfp (see Figs. 1 and 2). All values are normalised to the expression from the promoter less reporter vector, pSUN202 (negative control) and the GFP intensity is shown as relative intensity compared to the negative control. All measurements however were performed in triplicates. In situ localization of hupSL transcript To investigate selleck compound if the truncated parts of the hupSL promoter, except from being important for the expression levels, also affected the cellular localization of hupSL transcription fluorescence

microscopy was used to view the living cells. Furthermore, this study was carried out to analyze if the high transcription level of the shortest promoter fragment (construct E, promoter fragment stretching from -57 to tsp) was the result of a general low expression in all cells rather than high specific expression in the heterocyst. Images of the filaments were taken using bright field and fluorescence microscopy and then merging the images. The micrographs showed that promoter fragments A-D had heterocyst specific expression (Fig. 5). Surprisingly, even the shortest promoter construct E showed a heterocyst specific expression (Fig. 5). The promoter region of PrbcL fused to gfp, used as a positive control, gave, as expected, high expression primarily in vegetative cells [49, 50] (Fig. 5). Figure 5 In situ localization of hupSL transcript. Micrographs showing localization of the GFP expression from the hupSL promoter in nitrogen fixing filaments of Nostoc punctiforme ATCC 29133. N. punctiforme cells were transformed with a self replicative vector, pSUN202, containing deletions of the hupSL promoter fused to gfp (see Fig. 1).

Thus, precedents from other systems support our findings that spe

Thus, precedents from other systems support our findings that spectrin, adducin, and p4.1 can act independently during bacterial pathogenesis. Conclusions Invasion of intestinal epithelial cells and comet tail-based motility in host cells are key for S. flexneri to access replicative niches and disseminate throughout host tissues [2]. Here we have demonstrated that the actin-rich structures generated by these microbes also employ another cytoskeletal system, the spectrin cytoskeleton. Our identification of this structural network at these sites further highlights the importance of this system in bacterial pathogenesis and

indicates that these crucial segments in the pathogenesis of S. flexneri require a hybrid cytoskeletal meshwork, previously thought to be exclusive www.selleckchem.com/products/gsk2126458.html to actin. Methods Cells, bacteria and growth conditions HeLa cells (ATCC) were grown on #1 cover slips in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The bacterial strain utilized was S. flexneri (strain M90T). Bacteria were grown in standard trypticase soy. Infections HeLa cells were grown to approximately 70% confluency prior to infections. S. flexneri were grown overnight in standing culture, then diluted 80×, followed by growth in shaking

selleck culture at 37°C for 2.5 hours (OD600 nm = 0.6) after which 400 μl of the culture was added to the cells with 200 μl of growth media [31]. Infections were initiated by centrifugation for 10 mins at 700 g and 21°C. To www.selleckchem.com/products/SB-202190.html quantify invasion events, investigate initial tail formation and study comet tails, total infection times consisted of 0.5, 2.5 and 4.5 hours respectively. For classical invasion assays, cells were washed 2× with PBS after 20 minutes of infections and incubated in 100 ug/mL of gentamycin in 10% DMEM for 1 hour. Cells were washed 3× with PBS, lysed using 1% triton and plated for CFU counts. Invasion assays examined by microscopy

To quantify S. flexneri invasion, mafosfamide similar infection parameters were followed as in the classical invasion assay, however after 1 hour of gentmycin treatment the cells were washed with PBS three times prior to fixation and quantification of bacterial invasion via microscopy. Immunofluorescence Immunofluorescence procedures were performed as described previously [20]. Briefly, samples were fixed using 3% paraformaldehyde for 15 minutes then permeabilized using 0.1% Triton X-100 in PBS (without calcium or magnesium) (Hyclone) for 5 minutes. Prior to primary antibody treatments, samples were blocked in 5% normal goat serum in TPBS/0.1% BSA (0.05% Tween-20 and 0.1% BSA in PBS) for 20 minutes. Antibodies were then incubated on the cover slips overnight at 4°C. The next day secondary antibodies were applied for 1.5 hrs at 37°C. The cover slips were then mounted on glass slides using Prolong Gold with DAPI (Invitrogen).

One of our sequences affiliated with Crenarchaea cluster 1 1b, wh

One of our sequences affiliated with Crenarchaea cluster 1.1b, which includes several putative AOA [54–56]. However, it has recently been shown that not all amoA-carrying Thaumarchaeota are ammonia-oxidizing autotrophs [57]. The presence

of AOA at the Rya WWTP can therefore not be confirmed, and as has been suggested for other WWTPs [14, 16], AOA are most likely of minor or no importance for ammonia-oxidation at the Rya WWTP. One clone affiliated with Crenarchaea cluster 1.3. There are no cultured representatives of cluster 1.3, but spatial co-localization [58] and a relation between the abundance of cluster 1.3 and Methanosaeta-like species has been reported [42]. In other aggregate structures, such as anaerobic sludge Adriamycin mouse PI3K Inhibitor Library granules, Methanosaeta are important for structure and stability and they form dense aggregates which act as nuclei for granule formation [20]. In the activated sludge the Methanosaeta did not appear to have this function as they were mostly detected as small colonies or Mocetinostat solubility dmso single cells (Figure  11) and there was no apparent difference

in structure between flocs with high and low numbers of Methanosaeta. The lowest relative abundances of the Methanosaeta-like TRFs were observed in January and February 2004 (Figures  7 and 8). In October 2003 the two main Methanosaeta TRFs also decreased in relative abundance but it cannot be ruled out that the TRFs that appeared in those samples were also Methanosaeta (Table 4). The lowest water temperatures of the period were recorded during January and February 2004, which could have

reduced the survival or proliferation of Methanosaeta-like species and allowed other Archaea to increase. In anaerobic sludge, a decrease in Methanosaeta abundance has Adenosine been linked to granule disintegration [18, 19]. Although the flocs had high shear sensitivity and a more open structure in January and February 2004 when the Methanosaeta TRFs decreased and although there was a significant negative correlation between Methanosaeta TRFs and effluent non-settleable solids (Table 6) it cannot be concluded that the Archaea are important for the floc structure. The increased shear sensitivity and changed floc structure in January and February 2004 could be due to the reduced general microbial activity, which has been shown to decrease floc stability [5]. Furthermore, increased shear sensitivity and changed floc structure was also observed from June to August 2004, after the primary settlers were bypassed, but during this period the relative abundance of the Methanosaeta TRFs was 100%. Thus, if the composition of the Archaea community has any effect on floc structure or stability it is certainly only one of many other factors. Conclusions By sequencing and T-RFLP analysis of 16S rRNA genes and FISH we showed that Archaea were present in the activated sludge of a full-scale WWTP.

0) or exchanged by repetitive concentration/dilution using 30 kDa

0) or exchanged by repetitive concentration/dilution using 30 kDa Centricon or Microcon filters into 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5 or (2-[N-cyclohexylamino]ethane sulfonic acid (CHES) pH 9.5. Finally, the samples were concentrated to an OD802 of 80–130. CW X-band EPR measurements were performed with a Bruker ESP 300 spectrometer at room temperature using a rectangular cavity

with optical access (TE102, ER 4102ST, Bruker), using a capillary with 1 mm inner diameter. The radical cation P•+ was created via continuous illumination with white light in situ, using heat-absorbing glass and water filters. CW X-band Special TRIPLE measurements were done on the same spectrometer at 288 K. A home-built ENDOR cavity was used, similar to the one previously described (Zweygart et al. 1994),

but with a nitrogen gas cooling system. The cation radical P•+ was created in situ as described above. The data analysis was performed selleck compound using home-written routines in Matlab™, similar to the program used before (Tränkle and Lendzian 1989). In several cases, a baseline was recorded under identical conditions (with the magnetic field off-resonant and subtracted) under the assumption that possible drifts and artifacts would be the same in both cases. Q-band EPR and ENDOR measurements in frozen solution were done on a Bruker Elexsys E580 spectrometer at 80 K. For frozen solution experiments, sucrose (60%) was added to all samples. A home-built resonator was used (Silakov et al. 2007), similar to the one described previously SBI-0206965 order (Sienkiewicz et al. 1996). A Davies-type pulse ENDOR experiment (Davies 1974) was performed as described previously (Epel et al. 2006). Results X-band EPR measurements Measurements using the X-band EPR spectrometer were performed for both wild-type RCs and the four mutants, ND(L170), HE(L168), ND(M199), and HE(L168)/ND(L170), in liquid solution. In all cases, the spectrum was a single unresolved line centered at g

close to g e (see Fig. 2 for an example). Fig. 2 Comparison of CW X-band EPR spectra of light-induced P•+ in RCs from Rb. sphaeroides wild type with hepta-histidine tag (WT-H7) (red line) and from ND(L170) (blue line) at pH 8.0 For wild-type RCs at pH 8.0, the spectrum was simulated using a Gaussian before function with a linewidth ΔB pp (peak-to-peak) of 9.6 G (±0.2 G) at g = 2.0026 in agreement with published data of this radical in RCs from Rb. sphaeroides 2.4.1 (see for example Feher et al. 1975; Norris et al. 1971; Artz et al. 1997). The spectrum of the four mutant RCs at pH 8.0 were fitted yielding the same selleck chemicals llc g-value and different Gaussian linewidths. For all of the mutants, the EPR linewidth was increased relative to wild type. The linewidth is smallest for the ND(M199) mutant (10.1 G), followed by the HE(L168) mutant (10.2 G), with the ND(L170) mutant and the double mutant HE(L168)/ND(L170) having the most pronounced increase (11.0 G).

Infect Immun 1983,41(3):1212–1216 PubMed 12 Paton JC, Rowan-Kell

DNA Damage inhibitor Infect Immun 1983,41(3):1212–1216.PubMed 12. Paton JC, Rowan-Kelly B, Ferrante A: Activation of human complement by the pneumococcal toxin pneumolysin. Infect Immun 1984,43(3):1085–1087.PubMed 13. Boulnois GJ, Paton JC, Mitchell TJ, Andrew PW: Structure and function of pneumolysin, the multifunctional, thiol-activated

toxin of Streptococcus pneumoniae. Mol Microbiol 1991,5(11):2611–2616.PubMedCrossRef 14. Hammerschmidt S, Bethe G, Remane PH, Chhatwal GS: Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae. Infect Immun 1999,67(4):1683–1687.PubMed 15. Janulczyk R, Iannelli F, Sjoholm AG, Pozzi G, Bjorck L: Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function. J Biol Chem 2000,275(47):37257–37263.PubMedCrossRef 16.

Romanello V, Marcacci M, Dal Molin F, Moschioni buy AZD9291 M, Censini S, Covacci A, Baritussio AG, Montecucco C, Tonello F: Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease. Protein Expr Purif 2006,45(1):142–149.PubMedCrossRef 17. King SJ, Hippe KR, Gould JM, Bae D, Peterson S, Cline RT, Fasching C, Janoff EN, Weiser JN: Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in MLN2238 vivo and protect the airway. Mol Microbiol 2004,54(1):159–171.PubMedCrossRef 18. Holmes AR, McNab R, Millsap KW, Rohde M, Hammerschmidt S, Mawdsley JL, Jenkinson HF: The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol 2001,41(6):1395–1408.PubMedCrossRef 19. Zhang JR, Mostov KE, Lamm ME, Nanno M, Shimida S, Ohwaki M, Tuomanen E: The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. PLEK2 Cell 2000,102(6):827–837.PubMedCrossRef 20. Anderton JM, Rajam G, Romero-Steiner S, Summer S, Kowalczyk AP, Carlone GM, Sampson JS, Ades EW: E-cadherin is a receptor for the common protein

pneumococcal surface adhesin A (PsaA) of Streptococcus pneumoniae. Microb Pathog 2007,42(5–6):225–236.PubMedCrossRef 21. Lu L, Ma Y, Zhang JR: Streptococcus pneumoniae recruits complement factor H through the amino terminus of CbpA. J Biol Chem 2006,281(22):15464–15474.PubMedCrossRef 22. Hammerschmidt S, Tillig MP, Wolff S, Vaerman JP, Chhatwal GS: Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif. Mol Microbiol 2000,36(3):726–736.PubMedCrossRef 23. Bergmann S, Rohde M, Chhatwal GS, Hammerschmidt S: alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol 2001,40(6):1273–1287.PubMedCrossRef 24. Bergmann S, Rohde M, Hammerschmidt S: Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein. Infect Immun 2004,72(4):2416–2419.PubMedCrossRef 25.

Also, minor errors (any false

Also, minor errors (any false VX-680 concentration result involving an intermediate result), major errors (false-resistant results) and very major errors (false-susceptible results) were calculated. Statistical analysis Bacterial load in ID broth for GPC and GNR was compared using an independent samples t-test. Results Inoculum of bacteria in ID broth after use of serum separator tubes (SSTs) In total, 134 blood cultures were included,

from 116 patients. The inoculum of GPC in ID broth was on average 3.6 × 107 CFU/ml, whereas that of GNR was 1.8 × 108 CFU/ml, which was a TSA HDAC molecular weight significant difference (95% CI between -1.7 × 108 and -1.2 × 108; P < 0.001). ID of GNR with the direct Phoenix method NSC23766 cost ID with direct inoculation was correct for 95.2% of all tested Enterobacteriaceae. One Escherichia coli strain was incorrectly identified as Salmonella choleraesuis with the direct method. One Serratia marcescens strain could not be identified with the direct method. Identification for Pseudomonas spp. was correct in 71.4%. Both errors in this group involved strains of Pseudomonas aeruginosa that were incorrectly identified as Pseudomonas fluorescens (Table 1). No errors in ID were observed

for the routine method. Table 1 Results of identification of GNR with the direct method   Total no. of strains No. of unidentified strains No. of misidentified strains ID of misidentified strains Enterobacteriaceae         E. coli 26   1 Salmonella choleraesuis K. pneumoniae spp. pneumoniae 8       S. marcescens 4 1     K. oxytoca 1       P. mirabilis 1  

    E. cloacae 1       M. morganii 1       Non-fermenters         P. aeruginosa 7   2 Pseudomonas fluorescens Antibiotic susceptibility testing (AST) of GNR Results of AST were available for 49 strains, one P. aeruginosa strain failed to grow sufficiently in the Phoenix system so no results were available for the direct method. Categorical agreement of the direct method with results of the standard method for GNR was 97.6%. After discrepancy analysis of the results of AST, this percentage rose to 99.0%, with 5 minor errors (0.7%), no major errors, the and 2 very major errors (0.3%) (Table 2). Both very major errors occurred with trimethoprim-sulfamethoxazole in Pseudomonas aeruginosa strains. Categorical agreement of the standard method after discrepancy analysis was 98.4% (table 2). One very major error occurred with trimethoprim-sulfamethoxazole. No antibiotic showed a categorical agreement of <95% (Table 3). Table 2 Agreements and errors for AST of GPC and GNR for the direct and routinely used Phoenix method   Direct vs routinely used method Direct method after discrepancy analysis Routine method after discrepancy analysis GPC (n = 84)       Categorical agreement 93.1% 95.4% 97.3% Minor errors 1.7% 1.1% 0.7% Major errors 4.2% 3.1% 0.8% Very major errors 0.9% 0.4% 1.