coli control (Fig 5, lane 4) Twenty-five years after its charac

coli control (Fig. 5, lane 4). Twenty-five years after its characterization as an obligate intracellular Alphaproteobacteria (Fryer et al., 1992), it has only recently been demonstrated that P. salmonis selleck inhibitor is truly a free-living bacterial pathogen, belonging to the Gammaproteobacteria group (Fryer & Hedrick, 2003). The bacteria is known to survive in either fresh (Graggero et al., 1995) or marine waters (Olivares & Marshall, 2010) and moreover it is also known

to be highly adaptable when exposed to limiting and/or stressing conditions, which mimics its natural situation in the oceans (Rojas et al., 2008). Additionally, the presence of insertion sequences and putatively other mobile genetic elements in P. salmonis represents a solid evidence that the adaptability potential of the bacteria resides in its versatile genome (Marshall et al., 2011). In this context, the description of a TA locus in P. salmonis appears to be a natural consequence of this versatility. Indeed, TA loci are conserved (often in multiple copies) in the genomes of many organisms that can cause persistent infections and/or persist in the environment: M. tuberculosis, Helicobacter pylori, Coxiella burnetii, Leptospira interrogans, Vibrio cholerae, Ridaforolimus order and Salmonella

enterica serovars Typhi and Typhimurium, as well as Haemophilus influenzae, are good examples of this fact (Daines et al., 2007). Additionally, it is important to consider that TA loci are highly abundant in free-living bacteria, but

lost from host-associated microorganisms (Pandey & Gerdes, 2005). To date, nine TA families have been reported in the literature: VapBC, RelE, ParE, MAzF, Doc, HipA, HigB, CcdB, and ω-ɛ-ζ (Van Melderen & Saavedra De Bast, 2009). The VapBC is the largest family of bacterial TA modules, representing close to 40% of all the TA loci known, and grouped together by virtue of their toxin components, in most cases belong to the PilT N-terminal domain family of proteins, which in turn function as ribonucleases (Cooper et al., 2009; Robson et al., 2009). Thus, it appears logical and important to identify TA loci in emerging Tobramycin prokaryotic organisms in order to improve our understanding of these systems, and more broadly, in attempting to understand the cellular mechanisms behind bacterial adaptation (Sevin & Barloy-Hubler, 2007). We have characterized a new and functional bicistronic operon that encodes the two genes of a Type II TA module in P. salmonis. The organization of the P. salmonis TA locus shows many characteristics of other bacterial TA modules. The presence of IRs in the promoter region (Fig. 1) is a feature that is present in various Type II TA systems, such as the vapBC and ChpK operons of L. interrogans (Picardeau et al., 2001; Zhang et al., 2004). The localization of the antitoxin gene upstream of the toxin ORF is a distinctive feature shared by all Type II TA loci homologous to the P. salmonis system. The P.

1 μg L−1) This ability remained stable after the fungus was cult

1 μg L−1). This ability remained stable after the fungus was cultured for five generations. The other three ts PCR positive isolates only produced traces of taxol. The isolate SBU-16 (Fig. 4) was identified based on its morphological characteristics as well as ITS rDNA gene sequencing. Colonies on PCA are effuse, pale brown, and do not sporulate abundantly. The mycelium is septate and pale brown. Conidiophores are solitary, occasionally short-branched, pale brown to brown, smooth, 1–4-septate, 14–110 × 3–5.0 μm, cylindrical,

and at the apex swollen to 6–8 μm. Conidia develop singly and almost entirely through a narrow pore at the apex of each conidiophore, medium brown, oblong to oblong-ellipsoid, subtruncate at the apex, rounded or subtruncate at the base, straight or slightly curved, with 1–3 AG-014699 chemical structure transverse septa, and usually distinctly constricted in the

middle, 0–3 longitudinal or oblique septa, 15–30 × 12–18 μm (av. 21.84 × 14.06 μm), L/W ratio is 1.4–2.16 (av. 2.0) dark, and thin-walled. Ascomata develop in large numbers within PCA and PDA and on the firm base of an alfalfa stem on the PCA, but they contain immature asci (Fig. 4a). Isolate SBU-16 exhibits the key morphological characters of Stemphylium, including the proliferation and swollen apical cell or region of the conidiophores (Simmons, 1967, 1969) as well as morphological characters of Stemphylium sedicola (Simmons, 2001). Percurrently proliferating conidiophores are recognized as the principal morphological characteristic that clearly distinguishes Stemphylium from two similar genera, Ulocladium and Palbociclib Alternaria (Wang et al., 2010). Although the

identification of Stemphylium species is based principally on morphological characteristics of conidium and conidiophores, many of these characters often overlap among species, making species determinations difficult (Leach & Aragaki, 1970). In addition, the systematic position of the isolate Cediranib (AZD2171) SBU-16 was estimated by a sequence comparison of the ITS region with other species of the genus Stemphylium from the GenBank. Sequences of the SBU-16 in the ITS region were 530 bp. An online blast search of the ITS gene sequence of the SBU-16 isolate exhibited 99% similarity with several species of the genus Stemphylium and uncultured endophytic fungi. Evolutionary distances were calculated for a dataset that consisted of the sequences of the SBU-16 isolate and other species of the genus Stemphylium. The ITS neighbor-joining tree (Fig. 5) was reconstructed on the basis of the obtained distance matrix data. Finally, according to the evolutionary distance and morphological characters, isolate SBU-16 was identified as S. sedicola SBU-16. DNA sequence data are now commonly used to test morphological concepts and other taxonomic hypotheses (Hunter et al., 2006). The ITS DNA sequence is a widely accepted DNA marker for identifying fungi (Nguyen & Seifert, 2008).

A convenience sample of 60 customers were approached in a communi

A convenience sample of 60 customers were approached in a community pharmacy (60% male, 62% White, 38% of minority ethnic origin aged 18–65), to complete a face-to-face questionnaire. Participants were asked to select mutually exclusive responses reflecting their initial reaction to a fictitious pilot version of a vignette in which Dr Wilson does not prescribe

an antibiotic for David, an adult patient who had a cold and sore throat for the last five days. She recommended that he should visit a pharmacy for minor ailment advice. Participants were invited to provide an ‘open-text’ explanation CHIR-99021 molecular weight why antibiotics were not prescribed. The method was developed by a pharmacy student (MA) and pre-piloted with academic staff at a school of pharmacy and two general medical practitioners. The AZD2014 price question stem and response options

(to Dr Wilson’s decision) are shown in Table 1 and data were analysed using SPSS, Version 20. Ethical approval was granted by a Faculty Research Ethics Committee. Sixteen (27%) respondents disagreed with Dr Wilson’s decision and 5 of these thought Dr Wilson had incorrectly assumed antibiotics would not work (Table 1). Although 44 (73%) agreed with Dr Wilson’s decision, over a quarter (12) of this group felt frustrated with the outcome. The vignette may provide a basis for identifying lay misconceptions of reasons for restricting antibiotic prescribing such as – “[to] prevent the body becoming immune to it” and “if you keep taking the same medication it becomes ineffective” and – “…you should give it a rest and get the old bacteria out of the system”. These findings show that there is potential for the pilot vignette to identify an initial (emotional) response to a doctor’s decision not to prescribe antibiotics and to gauge opinion regarding the rationale to refuse

antibiotics. This method requires further testing in order to establish its validity and should be repeated in a larger representative sample of adults in order to establish whether these trends are generalizable. 1. Hoffman D, Botha J and Kleinschmidt I (2003). An assessment of factors influencing the prescribing of antibiotics in acute respiratory illness: a questionnaire study. SA Fam Pract; 45(6) 22–24. K. Kumalo, A. Gomes, G. Calabrese, R. Kayyali, S. Nabhani Kingston University, Non-specific serine/threonine protein kinase London, UK The aim of this study was to seek the perception of the public in relation to the safety and use of e-cigarettes. E-cigarettes were perceived to be ‘very safe’ mainly by smokers (96%) and ‘very unsafe’ by non-smokers (62.5%). Thirty-eight per cent of the population were e-cigarettes users; with ‘alternative to smoking’ (21%), ‘can use them indoors’(19%), ‘help quit smoking’ (14%) being the most popular reasons for using e-cigarettes. There is limited amount of information available to the public. E-cigarettes are perceived to possess a reduced risk; however this depends on the smoking status.

Six reference lines were measured on the study cast: D + E space,

Six reference lines were measured on the study cast: D + E space, arch width, arch length, intercanine width, intercanine length, and arch perimeter. For each participant, the D + E space of the contralateral intact primary molar served as a control. A paired t-test was used to compare the cast measurements between initial examination and 12-month follow-up. A t-test was used to compare D + E space changes with those of the control group. Results.  The D + E space of the extraction side after 12 months was significantly smaller than that of the control side (P < 0.05) and the initial D + E space (P < 0.05). A significantly

greater arch perimeter, intercanine width, and intercanine length were found after 12 months compared with the initial parameters. No significant differences were found, however, in arch width or arch length between the initial examination Selleck JQ1 and the 12-month follow-up examination (P > 0.05). Conclusions.  The 12-month space changes in the maxillary dental arch after premature loss of a primary maxillary first molar consist mainly of distal drift of the primary canine toward the extraction site. Mesial movement of permanent molars or tilting of the primary molars did not occur. An increased arch dimension was found especially in the anterior segment (intercanine width and length). There is no need for the use of space maintainers from the results in this study

in cases of premature loss of a primary first molar. “
“International Journal of Paediatric Dentistry 2010; 20: 347–352

Aim.  To investigate the prevalence of dental KU-60019 supplier fluorosis in children who had participated in an oral health programme between the ages 2–5 years, including fluoride tablets from the age of 2 years. Design.  The study group consisted of 135 10- to 11-year-old children who had participated in the programme, including parent education, tooth-brushing instruction and prescribed fluoride tablets aminophylline (0.25 mg NaF) (2–3 years: 1 tablet/day; 3–5 years: 2 tablets/day). The prevalence of dental fluorosis in the study group was compared with that in a nonintervention reference group consisting of 129 children of the same ages. The analysis was based on photos of the permanent maxillary front teeth using the Thylstrup & Fejerskov (TF) Index. Results.  No statistically significant difference in prevalence of dental fluorosis was seen between the two groups. Forty-three percent of the children in the study group and 38% in the reference group had fluorosis, the majority of a mild nature (TF-score 1). None had a TF score above 2. The pattern was the same after correction for parent reported intake of tablets at 3 and 5 years of age. Conclusion.  Introduction of fluoride tablets at the age of 2 years did not result in increased prevalence of dental fluorosis. “
“International Journal of Paediatric Dentistry 2012; 22: 92–99 Background.

For the nested PCR, the conditions were: pre-PCR, 94 °C for 2 min

For the nested PCR, the conditions were: pre-PCR, 94 °C for 2 min for denaturation, followed by 30 cycles Ceritinib molecular weight (94 °C for 15 s, 60 °C for 30 s and 72 °C for 2 min) and then a final extension at 72 °C for 7 min. It should be noted that, from the 11th cycle, the time of elongation increased by 5 s for each cycle. All samples underwent two PCRs followed by

a purification step of the nested product. The presence of amplicons was then confirmed by separation on a 1% agarose gel. The purification was performed using QIAprep Spin Miniprep Kit 50 (Qiagen). Sequencing was performed at Genome Quebec (McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada) using eight primers (Virco) covering the PR-RT genes. The sequences were analysed using sequencer 4.5 (Gene Code Software Corporation, Ann Arbor, MI, USA). Determination

of subtypes and analyses of drug resistance mutations were performed using the Virco algorithm (Virconet, The sequences were aligned with references representing all subtypes and circulating recombinant forms (CRFs) using clustal w version 1.83 [8], followed STA-9090 nmr by manual alignment using bioedit version (IBIS Biosciences, Carlsbad, CA, USA). Subtype references were selected from the Los Alamos National Library database for HIV-1 ( The phylogenetic tree was constructed with mega software version 4.1 (Biodesign Institute, Tempe, AZ, USA), using the Kimura two-parameter model (neighbour-joining method) and a bootstrap value of 500 replicates. The sequences that were included were the consensus sequence for the M group and study sequences (n=101). Statistical tests were performed using sas software version 9.1 (SAS Institute, Cary,

NC, USA). Some data for one patient were not available, Tyrosine-protein kinase BLK so analyses of age, sex and CD4 cell count were performed on 100 patients. Viral load (VL) and resistance prevalence analyses were performed on 101 patients. These variables are expressed as medians with interquartile ranges (IQRs). The prevalences were determined with a confidence interval (CI) of 95%. The percentage of patients with CD<200, between 200-350 and over 350 cells/μL was also calculated. Among the 101 subjects included in this study, 42 were enrolled at CESAC, 43 at HGT and 16 at HPG. Clinical data were lacking for one subject. Among the remaining 100 subjects, 76 were women and 24 men. The median (IQR) age was 35 (18–65) years, the median (IQR) viral load was 400 000 (225–19 000 000) HIV-1 RNA copies/mL and the median (IQR) CD4 count was 135 (1–585) cells/μL.

9A and C from the present data set obtained before SC inactivatio

9A and C from the present data set obtained before SC inactivation). Despite this difference between the two monkeys, we found that SC inactivation again strongly disrupted microsaccade directions in monkey J during the attention task. Moreover, such disruption was consistent with a repulsion of microsaccades away from the inactivated region, as we observed in monkey M. To illustrate this, Fig. 9A and B plots the results from monkey J for the pre-injection (A) and post-injection (B) cases when the cue was placed in the affected region of SC inactivation, and Fig. 9C and D shows the results for when the foil was in the affected region. As just

mentioned, DAPT in vivo pre-injection data in this monkey revealed that the initial cue-induced bias in microsaccade directions was first towards the foil (Fig. 9A and C, red curve) and then towards

the cue (Fig. 9A and C, blue curve). During SC inactivation and when the cue was in the affected region, this modulation was again abolished (Fig. 9B, left, blue curve); there was instead a strong and rapid (~140 ms after cue onset) initial bias away from the cued location (red arrow) and an check details increase in movements towards neither the cue nor foil (Fig. 9B, right, black curve). This initial bias away from the cued location and towards neither location occurred ~110 ms earlier than the earliest directional modulation peak observed in any direction without SC inactivation in this monkey (referenced by the magenta lines). When the foil was in the affected region (Fig. 9D), microsaccade directions were very similar to those in the pre-injection case (Fig. 9C), as in monkey M, except that there was again a strong and rapid (~110 ms) bias away from the affected region, which, in this

case, corresponded to the foil location (Fig. 9D, middle, red arrow). In addition, unlike monkey M, monkey J showed stronger repulsion away from the affected region to the ‘neither’ stimulus locations than towards the diametrically opposite stimulus location, and he did so for both cue and foil in the affected region. Thus, the net effect of SC inactivation in this monkey Methamphetamine was to reduce movements towards the affected region in favor of movements away from it (in this case, including the ‘neither’ locations, and not just the diametrically opposite location, as was the case in monkey M). The directional time course analyses of Figs 8 and 9 also revealed that, in both monkeys, microsaccades at other times relative to cue onset could still be directed towards the affected region of space after SC inactivation. In particular, microsaccades with longer latencies after cue onset, when the expected effects of attention shifts would have subsided, were not impaired. For example, as shown in in Fig.

Some destinations are associated with increased risks regardless

Some destinations are associated with increased risks regardless of age. To prevent travelers from contracting diarrhea, adequate measures should focus on specific subpopulations. Travelers’ diarrhea is one of the most predominant health AZD1208 supplier threats among international travelers.1–3 Some

reports estimate that 20% to 50% of people traveling from industrialized countries to developing countries experience travelers’ diarrhea.4–6 Although most cases are mild and self-limiting, associated morbidity can affect the traveler’s well-being.1,3 Moreover, an exotic pathogen could spread from endemic regions to other communities.7,8 Thus, preventing travelers’ diarrhea is an important public health issue. To reduce traveler’s diarrhea, realistic preventive measures should be established based on accurate epidemiological findings.8 Unfortunately, however, reliable

information is scarce, as most relevant research has been conducted without denominator data and results might therefore be biased.7,9–11 Ideally, specific subpopulations with increased risk of travelers’ diarrhea should be identified based on data covering all relevant travelers. The quarantine station at Narita International Airport is the largest this website quarantine office in Japan and is responsible for checking more than half of the international passengers from abroad. The aims of this study were to undertake a descriptive analysis of the epidemiology of travelers’ diarrhea many and to determine the factors associated with contracting this disorder. We estimated diarrhea incidence using Immigration Bureau data as the denominator and quarantine data as the numerator. Specifically, we retrospectively investigated the characteristics of passengers arriving with diarrhea in terms of age, sex, seasonality, and travel destination. Travelers’ diarrhea was defined as “the passage of three or more unformed stools per 24 hour period, with at least one passage accompanied

by symptoms of nausea, vomiting, abdominal cramps or pain, fever or blood in the stool”12,13 during or shortly after travel. Travel destination was arbitrarily defined as “the location of the departing airport of the aircraft arriving at Narita International Airport,” because we had no information on each traveler’s travel route. The study was conducted at quarantine station, Narita International Airport, approximately 60 km east of central Tokyo. In 2003, this was the 26th busiest passenger airport and third busiest freight hub worldwide. The airport has two separate terminal buildings, and each building has two different quarantine stations with health consultation rooms. All arriving passengers are requested to report any health problems during the previous 4 weeks. The station distributes questionnaires mainly to passengers from cholera- and shigella-endemic countries/areas. Upon visiting the health consultation rooms, quarantine doctors ask for a detailed travel history and perform a physical examination.

Ca2+ increased the efficacy of

tetronasin, as would be pr

Ca2+ increased the efficacy of

tetronasin, as would be predicted, but Na+ was almost as effective, despite the affinity of tetronasin for Na+ being < 5% that for Ca2+ Selleckchem FDA-approved Drug Library (Grandjean & Laszlo, 1983). In general, however, the effects of changing cation concentrations were relatively small and some could not be explained simply by the reported ion specificity of the ionophores. One possible cause of the small response was most likely the relatively small changes in concentration and therefore ionic gradient that were considered feasible, based on what might be achieved in vivo. The increase in [Na+] was only 26%, which would have a small effect on the transmembrane Na+ gradient. However, the change in [Ca2+] was substantial, a 2.6-fold increase, yet potentiation of tetronasin was still small. Several studies have been made previously, with some success, to apply the principle of cation enhancement of ionophores with ruminal bacteria and ruminal fermentation. Rumpler et al. (1986) found that adding Na+ to the diet of steers receiving monensin or lasalocid caused methane production to be decreased. This result was therefore consistent with the main mode of action of monensin as it is presently understood (Russell, 1987), but not with the K+/H+ exchange mechanism proposed for lasalocid (Schwingel et al., 1989). Increasing [K+] increased the potency of monensin towards ruminal bacteria in vitro

(Dawson & Boling, 1987), which

might not be expected to occur if the direction of induced K+ flux was outward, as in the Russell Trichostatin A (1987) scheme. Chirase et al. (1987) observed a significant interaction between K+ and lasalocid in continuous cultures, but also Mg2+ and monensin or lasalocid despite the low affinity of these ionophores for divalent ions. Thus, although interactions undoubtedly occur between the concentrations of individual cations and the efficacy of ionophores, their magnitude and direction do not always appear to correspond to known ionophore specificity tuclazepam and the magnitude and direction of transmembrane ion gradients that have been measured in ruminal bacteria. Furthermore, the effects of combinations of cations and ionophores appeared to be species dependent, possibly indicating that transmembrane ion gradients are different in different rumen bacterial species. The measurements of protonmotive force and ATP pools in E. ruminantium may help to explain some of these observations. Despite a rapid inhibition of cell growth, only relatively minor changes in intracellular cation concentrations were seen when monensin or tetronasin was added to the culture. Some efflux of Na+ and K+ was induced by monensin and Ca2+ by tetronasin. Undoubtedly, the measured ion concentrations in whole cells may not reflect the concentration of ions free in solution; cell walls, proteins and nucleic acids would be expected to bind Na+, K+ and Ca2+.

(1988) In addition, determination of yeast cultivation factors t

(1988). In addition, determination of yeast cultivation factors that can influence cell resistance to dehydration with concomitant reversible suspension of yeast metabolism has been reported previously.

For example, yeast cultivation in rich nutrient media has been shown to lead to the formation of more resistant OSI-906 cell line yeast populations compared with cells grown in poor synthetic nutrient media (Beker & Rapoport, 1987). Additionally, stationary-phase cells of bakers’ yeast, S. cerevisiae, are rather resistant to dehydration–rehydration, whereas the viability of exponential-phase cells following dehydration is severely compromised (Beker & Rapoport, 1987). It has been established that key metal ions, such as magnesium and calcium, play important roles in yeast physiology and biotechnology (Walker, 1994, 1999, 2004). Magnesium bioavailability dramatically influences yeast growth and metabolism in a beneficial manner, but calcium ions can antagonize essential magnesium-dependent functions in yeast (Walker, 1999). Sufficiency of intracellular free magnesium ions is absolutely required for the function of key enzymes and for ICG-001 cell membrane stabilization. Regarding the latter, magnesium acts in the physiological stress protection of yeast cells, by preventing increases in cell

membrane permeability caused by ethanol- and temperature-induced stress (Birch & Walker, 2000). The aim of the present investigation was to determine whether magnesium and calcium ions influenced the resistance of yeast cells to dehydration–rehydration. old Cultures of the yeast S. cerevisiae strain 14 used in this work were received from the collection of the Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia. Cultures were grown on nutrient media containing (g L−1): molasses, 20; (NH4)2SO4, 3.7; MgSO4, 0.75; NaCl, 0.5; KH2PO4, 1.0;

K2HPO4, 0.13, pH 5.0; in flasks with total volume 250 mL in an orbital shaker (140 r.p.m.) at 30 °C. In some experiments, the nutrient medium did not contain MgSO4 or contained its higher concentration – 1.5 g L−1. In Ca2+-supplementation experiments, calcium salts were added to the medium in concentrations of 2.0 or 5.0 g L−1. Biomass yield was determined by its drying to a constant weight at 105 °C. Biomass dehydration was performed using a convective method in an oven at 30 °C for 24 h. The residual moisture reached in these conditions was 8–10%, determined by drying to a constant weight at 105 °C. At such residual moisture (if adequately dehydrated), yeast can maintain its viability due to being in a state of anhydrobiosis. The survival rates of dehydrated cultures were determined using fluorescence microscopy with the fluorochrome primulin. We have previously shown that, using certain conditions for yeast dehydration, this viability test corresponds very well to traditional tests based on agar plate counts (Rapoport & Meysel, 1985).

[35] The completion of TABS involves the patient answering two se

[35] The completion of TABS involves the patient answering two sets of four questions using a five-point Likert scale. Answers from the first set give an adherence behaviour score (ABS) and answers from the second set give a non-adherence behaviour score (NABS). ABS of less than 19 out of 20 denote a lack of adherence behaviour whereas NABS of more than eight out of 20 can be defined as non-adherence behaviour.[35] Low scores for ABS are

suggestive of intentional non-adherence whereas low scores for question 5 (NABS) suggest unintentional non-adherence. It should be noted that although TABS comprises ABS and NABS, cumulative totals of the scores are unable to be given due to the inverse relationship between the scoring for ABS and NABS. Descriptive statistics were used to characterise the sample. A semi-structured Deforolimus molecular weight interview was selected as the most appropriate methodology to explore patients’ ideas, concerns and expectations about adherence to medication. Interviews were conducted by the corresponding author over a 7-week period from May 2010. The topic guide for semi-structured interviews CRM1 inhibitor was adapted from another study of medication adherence in patients with chronic illness.[22] This was reviewed for appropriateness

by the team prior to use. All interviews were digitally recorded before being transcribed by cardiology secretarial staff. Once the interviews were transcribed the accuracy of the transcriptions was scrutinised by the corresponding author. Patient confidentiality was maintained by omitting all names and identifiers, and patient approval for the use of direct quotations was obtained. Once the qualitative data had been transcribed CYTH4 the transcripts were loaded into computer-assisted qualitative data analysis software (Atlas.ti version 6.0.1; Atlas.ti GmbH, Berlin, Germany) which expedited analysis and enhanced ‘closeness’ with the data. A thematic framework was developed to code the transcripts. The original coding framework was agreed upon by GFR and SJL. GFR completed the coding

and SJL verified the accuracy of each applied code on all transcripts. Initially there was a process of familiarisation by listening/re-listening to the recorded interviews while reading/re-reading the transcripts, which allowed immersion in the data. A process of ‘coding’ was applied to the transcripts and these codes allowed for themes to be identified. The construction of the initial thematic framework was guided by the research aims and objectives and questions introduced to participants from the topic guides. However, the framework analysis could also be used to identify emergent themes expressed during the interviews, offering a unique flexibility to realise themes from outwith the topic guide. Contextual meaning for each quote and code were then indexed before being displayed in a process called charting.