One of these effectors corresponds

to SifA, a protein required for the formation of lysosomal glycoprotein (lgp)-containing structures (Sifs) in epithelial cells, which emerge from the vacuole (Stein et al., 1996; Boucrot et al., 2003). SifA binds to SseJ, host proteins SKIP (SifA kinesin-interacting protein), and RhoA family GTPases to cooperatively regulate the dynamics of SCV membrane in infected cells (Ohlson et al., 2008; Dumont et al., 2010). In addition, SopD2 corresponds to an SPI-2-regulated protein that promotes Sif SP600125 manufacturer formation, which contributes to virulence in mice (Ruiz-Albert et al., 2002; Brown et al., 2006; Schroeder et al., 2010). In S. Typhimurium, sopD2 encodes a protein sharing 42% identity with SopD, a known SPI-1-dependent Belnacasan price effector that plays a major role in gastroenteritis in animal models of Salmonella infection (Jones et al., 1998). Mutation of sopD2 in S. Typhimurium led to a prolonged survival in infected mice compared with

survival in mice infected with the otherwise isogenic wild-type strain. Furthermore, in a competition index assay, the sopD2 mutant was recovered at a significantly lower level compared with the wild type after the two strains coinfected the same mouse, indicating a significant role of this effector in Salmonella pathogenesis (Brumell et al., 2003). Salmonella enterica serovar Typhi lacks several effector proteins that in S. Typhimurium are crucial for the pathogenicity of the serovar (Raffatellu et al., 2005), such as sopD2, which in S. Typhi is described as a pseudogene (Parkhill et al., 2001; McClelland et al., 2004). We suggest that sopD2 inactivation is involved in human host adaptation of S. Typhi. To evaluate this, Rho in this study we examined the effect of trans-complementation

of S. Typhi with sopD2 from S. Typhimurium (sopD2STM) and its effect on reducing invasion of the epithelial cell line. Salmonella enterica serovar Typhi and S. Typhimurium strains used in this study are described in Table 1. Strains were routinely grown in Luria–Bertani (LB) at 37 °C with vigorous shaking, or anaerobically grown by adding 500 μL of sterile mineral oil as a barrier to oxygen before invasion assays in cultured human cells. When required, the medium was supplemented with chloramphenicol (20 μg mL−1). Comparative sequence analyses were made with the complete genome sequences of S. Typhi strains CT18 (AL513382) and Ty2 (AE014613), and the serovar Typhimurium (AE006468.1). The sequences were analyzed using blast, alignment and phylogeny tools available at http://www.ncbi.nlm.nih.gov/ and vector nt suite v.8 software (Invitrogen). PCR amplifications of S. Typhimurium 14028s sopD2 gene were performed using an Eppendorf thermal cycler and Taq DNA polymerase (Invitrogen). The reaction mixture contained 1 × PCR buffer, 1.

We OSI-744 concentration also assessed rates of CVD-related deaths by smoking status.

There were 192 CVD-related deaths in total. The adjusted IRR for current smokers compared with never smokers was 1.33 (951% CI 0.84, 2.10). The IRRs (95% CIs) for having stopped smoking for up to 1 year, 1–2 years, 2–3 years and >3 years were 0.90 (0.47,1.76), 0.59 (0.25,1.38), 1.20 (0.53,2.76) and 1.00 (0.47, 2.13), respectively. For the 20% of patients whose smoking status was not ever known, and who were therefore excluded from these analyses, the crude rates for CVD, CHD, MI and death were 4.1, 3.7, 2.6 and 18.8 per 1000 person-years, respectively. Of patients who reported current smoking status during follow-up, approximately 17% had some smoking data missing during follow-up. A sensitivity ATM signaling pathway analysis for both the CVD endpoints and mortality omitting all periods of follow-up where smoking status was missing yielded similar results (data not

shown). We assessed whether lipid, blood pressure and BMI levels changed in those patients who stopped smoking, and also whether there were changes in lipid- and blood pressure-lowering therapy. The median changes in total cholesterol, HDL-C, total cholesterol:HDL-C ratio, triglycerides, systolic and diastolic blood pressure and BMI were all zero up to 2 years following smoking cessation. There were, however, small mean decreases in total cholesterol [mean (standard deviation SD) –0.12 (1.16)], total cholesterol:HDL-C ratio [mean (SD) –0.32 (2.00)], triglycerides [mean (SD) –0.16 (2.03)] and BMI [mean (SD) –0.20 (1.55)], and small mean

increases in HDL-C [mean (SD) 0.04 (0.35)] and blood pressure [mean (SD) 0.40 (9.51) for diastolic and 1.48 (13.72) for systolic] at 2 years. The percentages of patients using lipid- and blood pressure-lowering medications both increased, from 12% and 9%, respectively, at the time of stopping smoking, to 19% and 13%, respectively, 2 years post smoking cessation. This is the first study to assess the impact of smoking cessation on CHD and mortality in an HIV-positive population. We found that the risk of MI, CVD and CHD decreased with each passing year of mafosfamide having stopped smoking, and after 3 years, the risk almost halved compared with the first year of stopping smoking. Rates of MI decreased from an almost fourfold increased relative risk compared with never smokers among patients in the first year of having stopped smoking to just over twofold greater relative risk among those who had stopped smoking >3 years previously. Although the reductions were less pronounced, the relative risk for CHD decreased from 2.5-fold to 1.8-fold, and that for CVD decreased from 2.3-fold to 1.5-fold.

aeruginosa is an obligate aerobe, it probably metabolizes drugs and repairs DNA in different ways to S. Typhimurium, a facultative anaerobe. Recently,

differences in DNA repair mechanisms were shown to have effects on the acquisition of drug resistance (Morero & Arqarana, 2009). Such difference Doxorubicin nmr in mutagen susceptibility suggests that NNN and BP may be capable of inducing drug resistance in microorganisms other than P. aeruginosa. MNU consistently conferred resistance to Rif and to CPFX resistance in P. aeruginosa. We further examined the effect of MNU concentration on the induction of resistance to these antibacterial agents. MNU concentration dependently increased Rif or CPFX resistance in P. aeruginosa, and the incidence of Rif resistance was 10 times higher than CPFX resistance. While we found nine mutations in rpoB that conferred Rif resistance,

only one mutation in gyrA, ACC to ATC at codon 83, conferred CPFX resistance to most of the CPFX-resistant strains of P. aeruginosa found in the experiment. Of the Rif-resistant P. aeruginosa induced by mutagens, check details 93% had mutations in the rpoB gene. The amino acid changes induced by mutagens were the same as those found in Rif-resistant M. tuberculosis (Murphy et al., 2006), a finding which suggests that mutagens may be implicated in the emergence of Rif-resistant M. tuberculosis. As described earlier, different species of bacteria may have different susceptibility to specific mutagens; thus, NNN and BP may be capable of conferring Rif resistance on M. tuberculosis. It would likely be fruitful to investigate the incidence of Rif-resistant M. tuberculosis between nonsmokers and smokers. Among CPFX-resistant P. aeruginosa induced by mutagens, 80% had mutations at codons Dichloromethane dehalogenase 83 and 87 in the gyrA gene, mutations that involve amino acid substitutions.

These same mutations are found in almost all the CPFX-resistant P. aeruginosa isolated from patients (Mouneimne et al., 1999; Akasaka et al., 2001). In 20% of mutagen-induced CPFX-resistant P. aeruginosa, we found no mutations in the quinolone resistance-determining region of gyrA. Consequently, we analyzed the entire gyrA sequence, however, we were unable to find any other mutations here. To further investigate what might confer CPFX resistance, we analyzed the gyrB, parC and parE genes and found mutations in gyrB and parE. All the mutations found in these genes would also lead to amino acid changes. Such mutations in gyrB or parE have not, however, been reported in clinically isolated CPFX resistant P. aeruginosa. In 11% of the samples of CPFX-resistant P. aeruginosa, we were unable to determine a likely cause for the resistance. Mutations in the regulatory genes for efflux pump proteins, resulting in an increased expression, have been reported to confer CPFX resistance (Higgins et al., 2003). Accordingly, we analyzed the sequence of regulatory genes nfxB and mexR, but found no mutations.

13 lens or 20 × 0.8 lens, in which case individual images were stitched together. Images of ß-galactosidase-immunostained sections for colocalization are single optical sections 0.8–0.9 μm thick, scanned selleck inhibitor with an LSM510, Axiovert 200M confocal microscope, acquired with a 20 × 0.75 lens or 40 × /1.4 oil-immersion lens. The scans were acquired sequentially to ensure that there was no carry over of signal between the channels. The images were quantified

using the ImageJ cell count plugin; data are averages of at least three sections from three mice each and shown as mean ± SEM % cells. Mice were briefly anesthetized with isoflurane inhalation and decapitated. The brains were dissected, GSK3235025 frozen in −40°C isopentane and stored at −80°C till used. Frozen brains were brought to −20°C; 14-μm-thick coronal cryostat sections collected on MMI membrane slides (Molecular Machines and Industries, Glattbrugg, Switzerland), dried for 1 h and stored at −80°C till used. For laser dissection, slides were stored on dry ice, one slide at a time was stained with 1 mL bis-benzimide (Hoechst 33258, Sanofi-Aventis;

10 μg/mL in 70% isopropanol) for 1 min, briefly rinsed in 70% isopropanol, dehydrated for 1 min in 100% isopropanol and air dried. The amount of material needed for simple immunoblotting Baf-A1 supplier using enhanced chemiluminescence detection can consist of 1000 cells or even fewer, depending on the abundance of the protein and the sensitivity of the primary antibody. Selected areas of the amygdala were cut using MMI CellCut laser microdissection system and captured on 0.5-mL caps of collecting tubes (MMI) for a final area of 200 000 μm2, sufficient for one or two determinations. Tissue areas were collected between Bregma −1.34 and −1.58

except for ITCs, which were collected over a larger range. Generally 10–14 cuts for the medial (mITCs) and lateral ITCs (lITCs), two cuts each for the lateral (no distinctions were made between the lateral and capsular subdivisions) and medial divisions of the CEA, and one cut each for LA and BA were sufficient. Care was taken that when multiple cuts were used they did not overlap on the cap. The tubes were put on dry ice and stored at −80 °C till used. Brief hematoxylin staining was also compatible with subsequent steps of immunoblotting. The slides could be recut at least a few times without noticeable deleterious effects. Immunoblotting was based on a previously described technique (Martinet et al., 2004), with some modifications.

, 2008). It is well known that stx2 play a key role in the development of HUS (Gyles, 2007). BTK inhibitor libraries In NSF O157, two different q genes,

q933 and q21, have been identified, giving evidence of higher production of stx2 in strains positive for q933 (LeJeune et al., 2004; Koitabashi et al., 2006; Matsumoto et al., 2008). Additionally, mutations in the stx2 promoter region have been observed in strains carrying the q21 gene, which probably also contribute to the reduced expression of stx2 (Matsumoto et al., 2008). However, the knowledge about the genomic regulation of stx2 expression in SF O157 is sparse. In the present study, the sequence upstream (including the q gene) and approximately 500-bp downstream of the stx2 gene in three Norwegian SF O157 isolates were sequenced, and a distinct q gene and different genes upstream of the stx2EDL933 gene, as compared to the NSF O157:H7 strain EDL933 (AE005174), were detected. The q gene and the genes upstream of stx2EDL933 in SF O157 had identical or similar sequence to the O111:H− strain 11128 (AP010960), a strain isolated from a patient with bloody diarrhoea in Japan in 2001 (Ogura et al.,

2007). stx-encoding lamboid bacteriophages show similarities in DNA sequences, yet they might be heterogeneous as evidenced by divergent gene organization Vorinostat datasheet and chromosomal location, as well as harbouring high degree of mosaic DNA structures (Unkmeir & Schmidt, 2000; Allison, 2007; Ogura et al., 2009). Based on these observations, our results indicate that the sequenced SF O157 isolates harboured different stx2EDL933-encoding phages than the NSF O157 strain EDL933 (Allison, 2007; Ogura et al., 2009). Furthermore, mosaic DNA structure was seen within the bacteriophage of strain 1108-2781 (FR874041), but not within the other two sequenced SF O157 strains, demonstrating that considerable diversity also exists among stx2EDL933-encoding bacteriophages within the group of SF O157. Two of 17 SF O157 strains were positive

for the stx8 primer set. Strain 1108-2781 (stx8+) had identical sequence with the NSF O157:H7 strain EDL933 in of this region, whereas strains 1106-4002 (FR874039) and 1109-0113 (FR874040) (both stx8−) showed identical sequence to the O111:H− strain 11128, thus explaining the PCR results. The stx8 primer set was suggested to differentiate NSF O157 into lineage I and II, where lineage I strains, positive for stx8, were shown to express more stx proteins and to have a higher pathogenic potential than the lineage II strains (stx8 negative) (Dowd & Williams, 2008). We did not investigate the expression of stx. However, one of the two stx8+ SF O157 isolates was obtained from a HUS patient, whereas as many as 80% (12/15) of the patients with SF O157 negative for stx8 developed HUS.

05). None of the LAB strains stimulated AFB1 accumulation in any of the fungal strains assayed. On the contrary, toxin production of A. flavus RC2053 and A. flavus RC2055 was totally inhibited by L. fermentum L23. It is likely that the low concentration of AFB1 in the presence of Lactobacillus strains could

be due to low mycelial biomass formation. Growth inhibition could directly affect AFB1 production as a result of low synthesis of the enzymes involved. Furthermore, AFB1 is a secondary metabolite that does not occur during primary growth of fungus, so that growth inhibition may reduce its production. In this study we have showed that there could exist a relationship between fungal growth and AFB1 production. In fact, these results showed that minimal yields of toxin coincided with ATM/ATR inhibitor clinical trial minimal mycelial growth. Tukey’s test of the data revealed the influence of L. fermentum L23 and L. rhamnosus L60 on growth parameters (lag phase and growth rate) and AFB1 production. Our results agree with Zinedine Selleck GSK1120212 et al. (2005), who demonstrated the ability of some strains of LAB to reduce the initial concentration of AFB1 in MRS broth.

Similar observations were made by Aryantha & Lunggani (2007), who observed that L. plantarum, L. fermentum and Lactobacillus delbrueckii significantly inhibited fungal growth of A. flavus and AFB1 production. Dalié et al. (2010) established that the main LAB recognized

for their ability to limit mycotoxinogenic mould growth belong to the genera Lactococcus and Lactobacillus, including L. rhamnosus, in agreement with our results. These results reflect a strong ability to inhibit growth rate and AFB1 production by both Lactobacillus strains with a wide spectrum of antimicrobial activity and high probiotic potential. This suggest that the use of LAB with antifungal properties instead of chemical preservatives would enable the food and feed industry to produce organic food without chemical additives. In addition to the known excellent properties of Lactobacillus strains, they could enhance Edoxaban the nutritional value and prolong the conservation of food. These results are important given that these aflatoxicogenic fungi are natural contaminants of raw materials used for food and feed production, which could be effectively controlled by L. rhamnosus L60 and L. fermentum L23, both strains having probiotic properties. It is concluded that, under favourable conditions, the two lactobacilli strains not only inhibited aflatoxicogenic fungal growth, but also inhibited AFB1 biosynthesis. Future studies with L. rhamnosus L60 and L. fermentum L23 may test the application of these lactobacilli as biocontrollers of fungal contaminants and also to extend the self life of food and feed stuffs, approaching in situ their probiotic properties.

However, increasing antibiotic resistance is threatening to undermine the effective treatment of shigellosis. Berberine is a natural isoquinoline alkaloid found in medicinal herbs, such as Rhizoma coptidis (Huanglian). Berberine Selleck FDA approved Drug Library has demonstrated a number of biological activities, including antisecretory, anti-inflammatory, antibacterial, antimalarial, antitumor and anticholesterol activities, and is widely used in the treatment of bacterial diarrhea and intestinal parasite infections in many countries (Yamamoto

et al., 1993; Iwasa et al., 1998). It has been shown that berberine has the properties of A–T base-specific DNA partial intercalation and generating singlet oxygen as a functional photosensitizer (Pilch et al., 1997; Brezova et al., 2004). Accumulated evidence suggests several mechanisms that may explain the antimicrobial activity of berberine. It has been reported that berberine interferes with the adherence of streptococci (Sun et al., 1988). It has also been demonstrated that berberine directly inhibits some Vibrio cholerae and Escherichia coli enterotoxins (Sack & Froehlich, 1982). In Leishmania donovani,

berberine exhibited an inhibitory action on macromolecular biosynthesis (Ghosh et al., 1985). Although berberine has been used in the treatment of gastrointestinal disorders, especially shigelleosis, for some time in Linsitinib supplier China (Chang, 1959), its effect on the causative agent of shigellosis is not yet well understood. Transcript profiling based on microarray technology enables us to investigate the response of the bacterial genome to antimicrobial agents, which provides useful clues to the mechanism of

action of the agents (Fu et al., 2007). In this study, whole-genome DNA microarray was used to examine transcriptional responses elicited by berberine in Shigella flexneri. Shigella flexneri 2a strain 301 (Sf301), our sequenced strain, was used in this study (Jin et al., 2002). The bacterium was grown at 37 °C with shaking (200 r.p.m.) on cation-adjusted Mueller–Hinton broth (caMHB), a medium recommended by the Clinical and Laboratory Standards Institute (CLSI) for susceptibility testing. Berberine chloride (BC) purchased from Sigma-Aldrich CYTH4 was resolved in dimethyl sulfoxide (DMSO) and diluted with caMHB. The minimal inhibitory concentration (MIC) of BC for Sf301 was determined according to the CLSI broth macrodilution methods for bacteria that grow aerobically (Clinical and Laboratory Standards Institute, 2006). Sf301, taken from a 24-h culture in caMHB, was inoculated in the same medium until reaching an OD600 nm of about 0.05. The cultures were then allowed to continue growing at 37 °C with shaking. When they were grown to early exponential growth phase (an OD600 nm of about 0.3), BC was added from the 400 × stock dissolved in DMSO into the cultures to give final concentrations of 160, 320, and 640 μg mL−1. A control with only DMSO was also included. The final DMSO concentration for all conditions was 1% v/v.

Putative transconjugants were confirmed by BOX-PCR typing. Profiles were generated by PCR amplification in 25 μL reaction mixtures containing 3.75 mm MgCl2, 0.2 mm dNTPs, 1× Stoffel buffer, 0.2 μm of primer BOX-AIR (5′-CTACGGCAAGGCGACGCTGACG-3′; Versalovic et al., 1991), 2.5 U Stoffel Taq polymerase (Applied Biosystems) and 1 μL of cell suspension prepared in 100 μL of distilled Epacadostat nmr water (~ 1.0 McFarland turbidity standard). Amplification was carried out as follows: initial denaturation for

7 min at 94 °C, then 35 cycles of denaturation at 94 °C for 7 min, followed by annealing at 53 °C for 1 min and extension at 65 °C for 8 min, and a final extension at 65 °C for 16 min. Generated profiles were separated in 1.5% agarose gels in 0.5× TBE buffer (50 mm Tris, 50 mm boric acid, 0.5 mm EDTA), at 50 V for 95 min, and stained with ethidium bromide. Plasmid DNA from donors and transconjugants was purified using Qiagen Plasmid Mini-kit (Qiagen GmbH, Germany). Diversity of plasmids was evaluated by plasmid restriction analysis using 5 U of PstI (CTGCAG) and 5 U of Bst1770I (GTATAC), according to the manufacturer’s instructions (Fermentas, Lithuania). Restriction patterns were visualized in 0.8% agarose gels. Electrophoresis was run at 40 V for 3 h in 0.5× TBE buffer and stained using ethidium bromide. Restriction

Y 27632 patterns were compared using GelCompar II software (Applied Maths, SintMartens-Latem, Belgium). Detection Farnesyltransferase of IncP-1, IncQ, IncN and IncW replicons and integrase genes was performed as previously described (Götz et al., 1996; Moura et al., 2010). Briefly, gels were transferred onto nylon membranes (Hybond-N, Amersham,

Germany) and hybridized in middle stringency conditions with PCR-derived specific digoxigenin-labelled probes for intI1, intI2, IncP-1 (trfA), IncQ (oriV), IncN (rep) and IncW (oriV) (Moura et al., 2010). Detection of IncA/C, IncB/O, IncF (FIA, FIB, FIC, FIIA, FrepB subgroups), IncHI1, IncHI2, IncI1-Iγ, IncK, IncL/M, IncU, IncT and IncY replicons was performed by PCR, using primers and conditions previously described (Carattoli et al., 2005). Results were confirmed by sequencing, except for IncFrep replicons, which were confirmed by Southern hybridization with digoxigenin-labelled probes generated by PCR from positive controls (Carattoli et al., 2005). The aim of this study was to evaluate the occurrence, diversity and conjugative potential of plasmids in integron-carrying bacteria from wastewater environments. The presence of plasmid DNA was confirmed in 77% (51 out of 66) of the strains. In the remaining 15 strains (~ 23%), no plasmids were detected by the plasmid extraction method used. Thus, most of the strains analysed harboured at least one plasmid, these strains being retrieved from all stages of the treatment process, including from final effluents (Table 1). Nevertheless, the presence of additional plasmids cannot be excluded.

The results imply close relations between LH motivational amplification functions and attention, and may inform our understanding of disorders in which motivational and attentional impairments co-occur. “
“The nuclei of the human amygdaloid complex can be distinguished from each other on the basis of their cytoarchitecture, DAPT price chemistry and connections, all of which process the information needed for the different functions (ranging from attention to memory and emotion) of the amygdala. This complex receives dopaminergic input that exerts modulatory

effects over its intrinsic network and is critical for reward-related learning and fear conditioning. To determine the specific distribution of the dopaminergic input through the different nuclei and

nuclear subdivisions of this structure we used stereological tools to quantify the fibers containing the dopamine transporter (used to signal the dopaminergic phenotype) in post-mortem samples from control individuals. Dopaminergic axons targeted every nucleus of the amygdaloid complex, and the density of dopamine transporter-containing axons varied considerably among its nuclear groups. The central group showed the greatest density of dopamine transporter-positive fibers, more than double the density of the basolateral group, the second most densely innervated structure. The dopamine transporter-positive innervation is very scant in the corticomedial group. The density of dopamine transporter-positive fibers did not vary among the nuclei of the basolateral group – i.e. basal, lateral and accessory basal nuclei – although there were significant density gradients among the subdivisions of these nuclei. Akt inhibitor These detailed quantitative data on dopamine transporter-positive innervation in the human amygdaloid complex

can offer a useful reference in future studies aimed at analysing putative dysfunctions of this system in diseases involving brain dopamine, such as certain anxiety disorders, Astemizole Parkinson′s disease and schizophrenia. “
“School of Biology, University of St. Andrews, Scotland, Fife, UK Exercise is known to have a strong effect on neuroproliferation in mammals ranging from rodents to humans. Recent studies have also shown that fatty acids and other dietary supplements can cause an upregulation of neurogenesis. It is not known, however, how exercise and diet interact in their effects on adult neurogenesis. We examined neuronal recruitment in multiple telencephalic sites in adult male European starlings (Sturnus vulgaris) exposed to a factorial combination of flight exercise, dietary fatty acids and antioxidants. Experimental birds were flown in a wind tunnel following a training regime that mimicked the bird’s natural flight behaviour. In addition to flight exercise, we manipulated the composition of dietary fatty acids and the level of enrichment with vitamin E, an antioxidant reported to enhance neuronal recruitment.

Participants were given an example of think-aloud interview technique and then asked to verbalize their thoughts

as they answered each question in the questionnaire and to indicate the reasons for providing the answers. Prompts (calendars, maps, and festival dates) were provided and on completion of the interview all participants were administered 24 structured follow-up probe questions. Use of prompts was observed and recorded. Scripted probes were used; responses were recorded by the investigator and subsequently analyzed. Items from the cognitive interviews were refined and incorporated into the final version of the questionnaire. We were not able to find copies (printed or electronic) of any questionnaires used in published travel-related Selleck Apitolisib studies, and none of the travel studies reported a process of validation. Thirty-four pooled items were selected for inclusion in the pre- and post-travel questionnaires (version 2). Sixty-four travelers were recruited to the prospective cohort study and completed the pre-travel questionnaire; the pilot study included 23 who had returned to complete the post-travel questionnaires. The remaining 38 travelers had not returned from travel and 3 were lost to follow-up. Age of the participants

ranged from 16 to 71 (median: 36) years, 42% were male, and 27% were overseas born. Most (62.5%) were tourists. Item-specific and general problems were identified by steps 3 and 4. Item-specific Crizotinib problems were mainly related to suboptimal clarity and an inadequate number of response categories provided. Table 1 provides examples of the item-specific problems identified, classification within the QAS framework, and the final revised why items. In addition, feedback by travelers, together with observed and self-reported difficulties in the pilot study, resulted in an expansion of the draft questionnaire items from 34 to 39. Seven of 19 post-travel

questionnaire items and 7 of 15 pre-travel questionnaire items were revised. Participants’ difficulties included deciding which destinations were “rural” locations and selection of appropriate traveler type category: definitions were therefore provided in the questionnaires. Some problems applied to multiple items across the questionnaire relating to QAS-99 categories of knowledge and memory. It was recognized that complicated travel itineraries and longer travel durations would be difficult to recall and record despite follow-up consultation within 2 weeks of return from travel. Open-ended questions were not selected for the categories of accommodation type or travel activities, as it was judged too difficult a recall task for travelers with long travel durations or complicated itineraries. Instead, a list of response options was provided. Some travelers did not report destination countries or health episodes in their correct temporal order.