Although there is solid evidence for the efficacy of in vivo and virtual reality (VR) exposure therapy for a specific phobia, there is a significant debate over whether techniques promoting distraction or relaxation have impairing or enhancing effects on treatment outcome. In the present pilot study, we investigated the effect of diaphragmatic breathing (DB) as a relaxation technique during VR exposure treatment.Twenty-nine patients with aviophobia were randomly assigned to VR exposure treatment either with or without diaphragmatic breathing (six cycles per minute). Subjective fear ratings, heart rate and skin conductance were assessed as indicators of fear during both the exposure and the test session one week later.The group that experienced VR exposure combined with diaphragmatic breathing showed a higher tendency to effectively overcome the fear of flying. Psychophysiological measures of fear decreased and self-efficacy increased in both groups with no significant difference between the groups.Our findings indicate that diaphragmatic breathing during VR exposure does not interfere with the treatment outcome and may even enhance treatment effects of VR exposure therapy for aviophobic patients.NCT02990208. Registered 07 December 2016.Retrospectively registered. ClinicalTrials.gov  Up to 40% of the population in industrialized countries suffers from a fear of flying; another 20% experiences strong fear during flying , 2. In oFor example, avoidance of fear-inducing situations or trying to distract oneself is considered, by some authors, to be an adaptive technique for anxious people reacting to threatening situations , 7. Othepanic self-efficacy and treatment effects on a follow-up test.Available evidence about the effects distraction on exposure treatment is inconclusive. In their literature review, Parrish et al.  come to In the present study, we aimed to investigate the role of relaxation as a coping strategy that could increase self-efficacy during treatment for a fear of flying. Relaxation was induced in this study through instructed diaphragmatic breathing (DB) during treatment. DB may be beneficial for exposure therapy since it reduces arousal on the physiological level  but at tFor the treatment protocol, we chose Virtual Reality Exposure Treatment (VRET) as it is easily controlled during the exposure and therefore optimal for assessing various process aspects of exposure therapy. In general, it has proven effective for the treatment of anxiety . ImportThe present study, designed as an inferiority trial, investigated the effect of diaphragmatic breathing as an additional coping strategy during VRET in patients with aviophobia. We assumed that DB would lead to less fear and physiological arousal during the VRET and to an enhanced treatment outcome. In our study, patients with aviophobia received treatment in VR with or without DB. Breathing techniques are often used in fear of flying courses, e.g. in Lufthansa’s seminars for relaxed flying . We assuThe aim of the study was to investigate the role of a relaxation technique, diaphragmatic breathing, as a coping mechanism during VR exposure therapy of aviophobia.From fifty-nine volunteers who had been recruited via advertisements displayed in public, only thirty  fulfilled the inclusion criteria . Exclusion criteria were pregnancy, heart disease and current involvement in psychotherapy and/or pharmacotherapy. One participant in the VRET group did not complete the treatment and was excluded from the statistical analyses.Socio-demographic variables and history of flight behavior are provided in Table In each of the groups, one participant did not fulfill all the criteria for aviophobia , a Head Mounted Display , and a personal computer. A tracking sensor  assessed the head movements and updated the field of vision of the HMD. Airplane sounds and security and breathing instructions were provided binaurally through headphones . The HMD refreshing rate was 60 Hz, the VR content and tracking latencies were in the millisecond range (<5 ms) .Flying phobia screening questionnaire (German version FSB  to the rest of the measurements .As we can see in Fig. . Furthermore, a significant interaction Time x Group  was evident. Follow-up t-tests of the within-subjects factor time revealed a significantly greater decrease of fear of flying from pre exposure  to the rest of the measurements .Excluding two participants older 55 years of age, there was still a significant main effect of time F2.67, 64.13 = 25.26, p < .001, ηp2 = .51). No effect involving the factor group was significant.As shown in Fig. F2.55, 56.04 = 23, 07, p < .001, ηp2 = .51). No effect involving the factor group was significant.Excluding the two participants aged above 55 there was a significant main effect of time as well , phase , and a significant Flight x Phase interaction . Group differences were not significant. To further analyze the Flight x Phase interaction, we conducted three separate repeated measures ANOVAs for the three flights. These ANOVAs revealed a significant main effect of phase in all flights . Further t-tests for the first flight showed that all pairs were significant (p < .05), except two pairs . In the second flight, all pairs reached significance (p < .05) and in the third flight all except c1/t2 (p = .861) did as well.Figure F1.22, 30.58 = 20.84, p = < .001, ηp2 = .46), phase , and a significant Flight x Phase interaction . Group differences were not significant. Repeated measures ANOVAs for the three flights revealed a significant main effect of phase in all flights .Excluding the two old participants, there were significant main effects of flight . In the second flight, all pairs reached significance as well (p < .05) and in the third flight all except c1/t2 (p = .862).Further F1.41, 38.05 = 17.13, p < .001, ηp2 = .39), phase , and a Flight x Phase interaction . No other effects were significant. We conducted three separate repeated measures ANOVAs for the three flights to follow up on the interaction. We conducted three separate repeated measures ANOVAs for the three flights to follow up on the interaction. These ANOVAs revealed a significant main effect of phase in all three flights . Further t-tests for the first flight showed that all pairs were significant (p < .05), except the pair (c1/t2: p = .512). In the second flight, all pairs reached significance (p < .05), except one pair that was marginally significant (c1/c2: p = .057). In the third flight, only 4 out of 6 pairs were significant .As demonstrated in Fig. F1.41, 35.30 = 15.92, p < .001, ηp2 = .39), phase , and a Flight x Phase interaction . No other effects were significant. We conducted three separate repeated measures ANOVAs for the three flights to follow up on the interaction. These ANOVAs revealed a significant main effect of phase in all three flights . Further t-tests for the first flight showed that all pairs were significant (p < .05), except the pair (c1/t2: p = .533). In the second flight, all pairs reached significance (p < .05), except one pair that was marginally significant (t1/t2: p = .055). In the third flight, only 4 out of 6 pairs were significant .Excluding the two older participants, an ANOVA revealed significant main effects of the factor flight  No other effects were significant. A follow-up analysis of the three flights separately showed a significant main effect of phase for the first and second flights , but not for the third flight (p = .384). Follow up t-tests showed that in the first flight all pairs except two  were significant. Similarly, in the second flight we could see the same pattern . This indicates that the turbulence manipulation was still influential within the first two flights but not in the third flight.As shown in Fig. F1.76, 44. 06 = 6.02, p = .007, ηp2 = .19) No other effects were significant. A follow-up analysis of the three flights separately showed a significant main effect of phase for the first and second flight , but not for the third flight (p = .493).Excluding the older participants, ANOVA revealed significant main effects of phase  were significant. Similarly, in the second flight we could see the same pattern .Follow up F1.27 = 45.29, p < .001, ηp2 = .63) and phase . Excluding the two participants aged > 55, there was an effect of flight  and phase .The fear ratings changed within flights—depending on the flight phase—and between flights. These changes are indicated by significant main effects of flight  and a Flight x Phase interaction . To follow up on the interaction, we conducted two ANOVAs, one for each flight with phase as the within subject factor. Only the first flight phase was marginally significant . The t-tests within the first phase showed a significant difference or marginally significant differences between t2 and c1 (p = .051), t1 (p = .038) and c2 (p = .068) and a marginally significant difference between t1 and c2 (p = .058).The HR decreased both within and between flights during the test flights. These patterns are partially reflected in the statistical analysis. The ANOVA revealed a significant main effect of flight (p = .074) but a Flight x Phase interaction . To follow up on the interaction, we conducted two ANOVAs, one for each flight with phase as the within subject factor. Neither for the first (p = .079) nor for the second (p = .104) flight there was a significant effect of phase.Excluding the two older participants, the ANOVA revealed no significant main effect of flight ). Excluding the two participants older than 55 there were no significant differences.The SCL remained relatively stable during the test flights. No significant differences were observed . However, there was no significant correlation between self-efficacy change and reduction in the FFS in either group.Table r = −.73, p = .007) and in the VRET + breathing group . Furthermore, there was a significant negative correlation between self-efficacy pre exposure and both the baseline FFS score  and the FFS score post test  in the VRET group. Furthermore, there was a significant correlation between the self-efficacy change and the FFS baseline score in the VRET + breathing group . Apart from that there were no significant correlations between self-efficacy  and the FFS scores (pre exposure and post test levels and FFS reduction).Excluding two participants older than 55 years of age there was a significant negative correlation between self-efficacy post test and the FFS score post test both in the VRET group (SD = 2.29), with a mean practice time of 8.67 min (SD = 4.43). Importantly, even without verbal instruction, patients continued to keep their breathing rhythm at 6 cycles per minute (mean breath duration was 9.88 (SD = 0.91) and 10.35 (SD = 2.1) in the exposure and test sessions, respectively).Descriptive analyses indicated that patients in the VRET + DB group were able to maintain the learned breathing frequency . Patients were instructed to practice diaphragmatic breathing between exposure and test sessions. According to their reports, patients practiced on average 4.93 times  of turbulence. A possible explanation for this result could be the course of the exposure session with turbulences during the flight and accelerations and decelerations during start and landing, which might have impeded or overshadowed habituation. Similarly, in a study on virtual reality exposure to heights which also involved repeated stressors (looking down) during exposure there was no habituation of the SCL either . SCL is Bandura  showed tn = 29) and therefore the power of our study is lowSome limitations to this study should be taken into account. First, there are different types of aviophobia , which wFinally, being a pilot study, the experiment was conducted with a limited number of participants. Nevertheless, our results indicated that DB may be a beneficial supplement for VR exposure therapy in aviophobics. Further research including larger samples will be needed in order to confirm this view.In conclusion, the present study provides additional evidence for the effectiveness of VR exposure therapy as a treatment for aviophobia. Importantly, our results indicate that using diaphragmatic breathing as an additional strategy to VR exposure could possibly lead to higher reductions in fear of flying compared to VR exposure alone. Further studies should analyze whether this effect is specific to aviophobia, or if it is encountered in other anxiety disorders  as well.


Campylobacter jejuni, possesses a periplasmic formate dehydrogenase and two terminal oxidases, which serve to metabolize formate and facilitate the use of oxygen as a terminal electron acceptor, respectively. Formate, a primary energy source for C. jejuni, inhibits oxidase activity in other bacteria. Here, we hypothesized that formate might affect both energy metabolism and microaerobic survival in C. jejuni. Subsequently, we showed that C. jejuni 81–176 (wildtype) exhibited enhanced chemoattraction to and respiration of formate in comparison to other organic acids. Formate also significantly increased C. jejuni’s growth, motility, and biofilm formation under microaerobic (5% O2) conditions. However, formate reduced oxidase activity under microaerobic conditions as well as aerotolerance and biofilm formation under ambient oxygen conditions. The expression of genes encoding the ribonucleotide reductase (RNR) and proteins that facilitate the use of alternative electron acceptors generally increased in the presence of formate. Taken together, formate might play a role in optimizing C. jejuni’s adaptation to the oxygen-limited gastrointestinal tract of the host. By affecting oxidase activity, formate possibly facilitates shuttling electrons to alternative acceptors, while likely conserving limited oxygen concentrations for other essential functions such as DNA synthesis via RNR which is required for C. jejuni’s growth.The foodborne microaerophilic pathogen,  Campylobacter jejuni, a foodborne pathogen, is associated with gastroenteritis and rare but serious post-infection complications in humansC. jejuni infections are commonly self-limiting, the ability of this pathogen to persist in food animals and to contaminate cognate food products poses a serious challenge for food safety and global healthC. jejuni exert a considerable economic burden. For example, in the USA, it was estimated that Campylobacter-associated infections cost $1.7 billion and result in the loss of 13,300 quality-adjusted life years (QALYs)C. jejuni, many factors that promote this pathogen’s ability to colonize and/ or infect the gastrointestinal (GI) tract of its hosts remain not completely understood.Salmonella invasion of the small intestine in miceSalmonella pathogenicity island 1 (SPI1) which encodes components of the type III secretion system and other proteins that play a role in the entry to eukaryotic cellsSalmonella invasionE. coliE. coli and Salmonella from killing by a cationic antimicrobial peptide, which was derived from the human bactericidal/permeability-increasing protein (BPI)C. jejuni’s pathobiology. For example, several studies reported that disparate C. jejuni mutant strains that were deficient in formate metabolism exhibited: (1)- reduced ability to infect human intestinal cells as well as primary chicken intestinal cells in vitro1012C. jejuni exhibits a strong chemoattraction towards formate14The mammalian GI tract is complex and encloses niches with diverse biological and physiochemical properties, including differences in pH (ranges between 4.0 and 8.5), hormones, enzymes, metabolites, and resident microbial communities. These factors can significantly influence the behavior and fate of a potential pathogen in the GI tract. For example, microbial metabolites such as short-chain fatty acids (SCFA), succinate, hydrogen, and secondary bile acids have been shown to affect the growth and virulence of gut pathogens55C. jejuni possesses a periplasmic formate dehydrogenase (FdhABCD), which facilitates the breakdown of formate into carbon dioxide and water1213bc1 complex, periplasmic c-type cytochromes, two terminal oxidases (CioAB: a cyanide-insensitive oxidase and CcoNOQP: a cb-type oxidase) and other respiratory proteins that facilitate the use of alternative terminal electron acceptors12C. jejuni to persist under low oxygen stress and maintain its microaerobic lifestyle1220C. jejuni, especially because an oxidase mutant was unable to colonize the GI tract of chickensC. jejuni and is considered an important respiratory acceptor in the chicken gutccoN::Cm strain) to colonize chickensC. jejuni under microaerobic (5% O2) and more restrictive oxygen conditions (<1% O2).C. jejuni 81–176 (wildtype)2, 10% CO2, 5% O2) or under <1% O2 (hereafter referred to as anaerobic conditions) which was generated using the BD GasPak Sachets system Campylobacter selective supplement , sodium formate, sodium succinate, sodium fumarate, sodium cyanide, sodium azide and sodium nitrate  were added to the MH medium when necessary.et al.C. jejuni was grown on laked horse-blood agar for 24 h. The cultures were then suspended in 10 mM phosphate buffer (pH 7.0), centrifuged, washed twice, and resuspended to achieve an OD600 of 0.7. The suspensions were transferred to the BIOLOG-AN plates (100 μL/well) which were then incubated microaerobically at 42 °C for 48 h and the OD550 was measured. Wells that did not contain any substrates were used as negative controls. The experiment was repeated three times.BIOLOG-AN plates  contain important metabolic substrates, and the metabolism of these substrates by bacteria results in the production of purple formazan which can be quantified spectrophotometrically at 550 nm. As described in Brandl et al.C. jejuni cultures were grown on MH agar plates under microaerobic conditions at 42 °C. The cultures were harvested, washed twice and suspended in Adler chemotaxis buffer [10 mM potassium phosphate and 0.1 mM EDTA (pH 7.0)] to achieve an OD600 of 0.02. One mL of the bacterial suspension was added on top of the agarose plugs containing the different chemicals. Changes in the OD600 of the bacterial suspension in response to the chemical gradient were quantified using a spectrophotometer. An agarose plug containing 1X PBS was used as a negative control and to assess chemotaxis-independent bacterial sedimentation. The samples were tested in triplicate and the experiment was repeated three times.Chemotaxis assay was performed as described in Zhulin C. jejuni’s growth, a formate solution (5 M) was freshly prepared by dissolving sodium formate in autoclaved and deionized water, which was followed by filtration using 0.2 μm sterile syringe filters . C. jejuni was then inoculated into MH broth and adjusted to an OD600 of 0.1, and formate  was immediately added to the cultures. Aliquots (180 μL) from each culture were transferred to a sterile 96-well plate, which was incubated with shaking (180 rpm) under microaerobic conditions at 42 °C. Growth was monitored at different time-points by measuring OD600600 between the cultures. For this purpose, the cultures were serially diluted (10-fold), which was followed by spreading aliquots (100 μL) on MH agar plates, and counting colony forming units (CFU).To evaluate the impact of formate on C. jejuni cultures were adjusted to OD600 of 0.02. Two μl of each culture were then stabbed into semisolid MH plates containing 0.4% agar and different concentrations of formate . The plates were incubated at 42 °C under microaerobic conditions. Diameters of the zones of motility were measured after 48 h of incubation.The impact of formate on motility and biofilm formation was determined as described previously11C. jejuni was suspended in MH broth containing formate  to achieve an OD600 of 0.05. One ml of culture was transferred to sterile borosilicate glass tubes, which were incubated for 72 h under microaerobic, anaerobic, and aerobic (ambient oxygen) conditions, respectively. The tubes were then gently washed with distilled water and stained with 0.1% crystal violet solution for 15 min. After further washing to remove excess stain, the tubes were left to dry at room temperature. The biofilms were then dissolved in 80% DMSO and quantified spectrophotometrically (OD570). The effect of different concentrations of sodium fumarate and sodium succinate on motility and biofilm formation was also assessed as described above. Fumarate and succinate were included as controls to contrast with the specific impact of formate on these phenotypes. All experiments were repeated at least three times and samples were tested in triplicate.Biofilm formation was assessed by using crystal violet staining11C. jejuni’s respiratory activity in the presence of formate was measured using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining as described elsewhere28C. jejuni cultures were adjusted to OD600 of 0.2 in MH broth. Different concentrations of formate were added to the cultures as described above. The cultures were then incubated shaking (200 rpm) under microaerobic conditions at 42 °C. After 30 min, 900 μL was aliquoted from each culture and 100 μL of CTC  was added. The samples were then mixed thoroughly and incubated in the dark at room temperature for 45 min. Colorimetric CTC reduction (respiration) was determined using a spectrophotometer (OD550). Respiratory activity was also conducted under anaerobic conditions in the presence and absence of formate and/or nitrate . The latter was added to assess respiration in the presence of an alternative electron acceptor. In order to reduce any compromise of the anaerobic conditions, these experiments were conducted in 96-well plates using 90 μL of culture and 10 μL of CTC for each reaction. In all experiments, sterile MH broth with CTC served as a negative control. Different concentrations of fumarate and succinate were included as additional controls to contrast with the specific impact of formate on respiration. The samples were tested in triplicate and the experiment was repeated three times.C. jejuni cultures were adjusted to OD600 of 0.2 in MH broth which was supplemented with formate as described above. After incubation for 30 min, 900 μL of each culture were used to assay for oxidase activity by adding 100 μL of TMPD solution, which was freshly prepared before each experiment. Alternatively, 1% ascorbic acid was added to the TMPD solution to reduce autooxidation570). The oxidase test was conducted also in the presence of sodium cyanide (100 μM) and sodium azide (250 μM)14Oxidase activity was measured using N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)C. jejuni 81–176 to aerobic (ambient oxygen) conditions in the presence of formate was assessed as described previously2731C. jejuni growing under microaerobic conditions on MH agar plates were harvested and suspended in MH broth to achieve an OD600 of 0.1. Cultures (100 mL) were transferred to different 250 mL sterile Erlenmeyer flasks covered with cotton plugs and were incubated under microaerobic conditions . Different concentrations of formate were added to each flask (t = 0 h post formate treatment) and the cultures were then incubated shaking (300 rpm) under ambient oxygen conditions at 42 °C. Aerotolerance was assessed by measuring OD600 using 1 mL plastic cuvettes and a spectrophotometer and counting CFU (as described above) at 8 h post formate treatment. Samples were tested in triplicate and the experiment was performed three times.The tolerance of C. jejuni cultures were incubated for 15 min with shaking (200 rpm) at 42 °C32(−ΔΔC(T)) methodThe expression of genes encoding respiratory proteins (electron donors and acceptors) and ribonucleotide reductase  followed by Tukey’s posttest. A C. jejuni’s respiration/ metabolism of these substrates was assessed using the BIOLOG-AN plates. Formate, succinate, fumarate, and aspartate were among the highly metabolized substrates (P < 0.05) in comparison to formate and fumarate , bstrates . In thesfumarate . The metfumarate .C. jejuni towards the aforementioned chemicals was evaluated. Under our experimental conditions, C. jejuni exhibited a significantly higher (P < 0.05) chemoattraction towards formate in comparison to all the other substrates (P < 0.05) in comparison to the other tested substrates  the growth (OD600) of C. jejuni .Supplementation of MH broth with formate significantly increased (. jejuni . However. jejuni . HoweverP < 0.05) C. jejuni’s zone of motility regardless of the added formate concentration (P < 0.05) only with relatively higher concentrations of succinate (50 mM) and fumarate (25 and 50 mM), respectively (Supplementation of semi-solid MH agar with formate significantly increased (ntration . In compectively . The mosectively .C. jejuni was associated with increased expression of the flagellar motility complexP < 0.05) in biofilm formation in response to the addition of formate (P > 0.05) in biofilm formation between C. jejuni cultures supplemented with 25 and 50 mM formate. In contrast, a significant increase in biofilm formation (P < 0.05) was only observed with the highest concentration of succinate (50 mM). The addition of 5 mM fumarate resulted in a significant increase (P < 0.05) in biofilm formation. However, there was no significant difference (P > 0.05) in biofilm formation between C. jejuni cultures supplemented with 5, 10, 25, and 50 mM fumarate.Biofilm formation in  formate . HoweverP < 0.05) in biofilm formation in response to the addition of formate (P > 0.05) in biofilm formation between C. jejuni cultures supplemented with either 5 and 10 mM formate or 25 and 50 mM formate. A significant decrease in biofilm formation (P < 0.05) was observed in response to 5 mM succinate and 5 mM fumarate, respectively. However, higher concentrations of succinate and fumarate did not significantly (P > 0.05) result in a further decrease in biofilm formation  in biofilm formation in response to the addition of formate (P > 0.05) in biofilm formation between C. jejuni cultures supplemented with 5 and 10 mM formate. A significant increase in biofilm formation (P < 0.05) was observed in response to 25 and 50 mM succinate. Biofilm formation was not significantly (P > 0.05) affected by any of the tested fumarate concentrations  respiration by ~4 times in comparison to cultures in MH broth only (0 mM formate added). The highest activity was observed with 5 mM formate (P < 0.05) in activity was observed with 50 mM formate in comparison to 5 mM formate. Regardless of the concentration, the respiratory activity in the presence of formate was significantly higher in comparison to succinate and fumarate (P < 0.05) the respiratory activity under anaerobic conditions but not under microaerobic conditions  decreased oxidase activity; however, the highest reduction was observed with 50 mM formate (P < 0.05) in oxidase activity occurred with 25 and 50 mM fumarate but not with other fumarate concentrations. Similar results were obtained when MH broth was replaced with MEMα (data not shown).It was reported that formate inhibits oxidase activity in some bacterial species20 formate . In comp formate . A signiP < 0.05) oxidase activity in comparison to cultures with formate, cyanide, or azide only  was significantly reduced after 8 h of exposure to ambient conditions . NeithernapA (nitrate reductase), which was downregulated  in response to formate in comparison to microaerobic conditions. Furthermore, the addition of formate and nitrate under anaerobic conditions significantly increased the expression of fdhA, cioA, napA, nrfA, and cjj81176_0403 in comparison to growth in a medium supplemented with formate only  is not entirely clear. Here, we hypothesized that physiological concentrations of formate simultaneously enhance respiration and reduce oxidase activity. This is a previously undescribed mechanism in C. jejuni that might shed a light on the adaptation of this pathogen to oxygen-limitation. We suggest that the formate-dependent reduction in oxidase activity activity . The latC. jejuni. We showed that formate is a strong chemoattractant and a preferred respiratory substrate  conditions. The latter is consistent with observations that a cioAB mutant did not have a significant defect in growth under 5% and 10% O2 in comparison to wildtypeccoN mutant was designated as O2-sensitive and could form colonies only under <7% oxygen conditionsC. sputorum subsp. bubulus, a microaerophilic bacterium, at low dissolved oxygen tensions but the growth slowed down at higher oxygen tensionsC. jejuni at higher oxygen conditions (>5% O2)14Physiological concentrations of formate significantly reduced oxidase activity of . jejuni . While tjejuni 20 and 6 unjejuni 20 under amjejuni 20. This incioA and ccoN in response to formate, especially under anaerobic conditions, was not expected. However, this might further indicate that the inhibition of oxidase activity by formate occurs on the enzymatic level. In a previous study, it was shown that formate binds to and inhibits cytochrome c oxidase in submitochondrial particlesnrdA increased in compensatory response to formate, which is crucial for DNA synthesis and the survival of C. jejuniC. jejuni in colonizing the gut.Our data suggested that the presence of formate can enhance the use alternative terminal electron acceptors, which is especially important in a niche that is oxygen limited and rich in anaerobic metabolites such as the gut. This was especially evident as the highest respiration was noted in the presence of formate and nitrate under anaerobic conditions which aligns with the expression data . The incC. jejuni, a bacterium with a relatively small genome, must possess the necessary adaptations and survival strategies to cope with and exploit this environment. Therefore, using a metabolite such as formate both as an energy generating substrate and a possible signal to optimize energy and survival constitutes an advantage and sheds a new insight on how C. jejuni interacts with its environment. It should be also noted that other gut metabolites might modulate the impact of formate, which further highlights the complexity of the interaction between a pathogen and its host’s gut.The gut is a complex niche(s), occupied by a diverse microbiota and metabolites and challenging conditions. How to cite this article: Kassem, I. I. et al. Formate simultaneously reduces oxidase activity and enhances respiration in Campylobacter jejuni. Sci. Rep.7, 40117; doi: 10.1038/srep40117 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, when comparing PTMCs with a unifocal diameter ≤1 cm vs. multifocal TTD >1 cm, significant differences were observed. In the Kaplan-Meier analysis, significant differences were observed between PTMCs with a unifocal diameter ≤1 cm vs. multifocal TTD >1 cm and multifocal TTD ≤1 cm vs. multifocal TTD >1 cm. Accordingly, TTD may represent a more accurate criterion for tumour size of PTCs and should be considered in the revised AJCC staging system.The current American Joint Committee (AJCC) on Cancer TNM classification does not describe the treatment of multifocal papillary thyroid microcarcinomas (PTMCs) with a total tumour diameter (TTD) >1 cm. Herein, we investigated this PTMC subgroup in terms of extrathyroidal extension (ETE), local infiltration, central lymph node metastasis (LNM), and prognosis. Consecutive patients (n = 1102) were identified and the proportions of LNM, ETE, and local infiltration were similar between PTCs with a unifocal tumour diameter >1 cm and ≤2 cm and PTMCs with a multifocal TTD >1 cm and ≤2 cm. The proportions of LNM, ETE, and local infiltration were also similar between PTMCs with a unifocal diameter ≤1 cm  PTMCs are diagnosed with increasing frequency. The proportion of PTMCs among all PTCs increased from 18.4% between 1983 and 1987 to 43.1% between 1998 and 2001 in France2, and similar results have been reported from other countries such as the United States and China5.According to the World Health Organization classification system, papillary thyroid microcarcinoma (PTMC) is defined as thyroid cancer measuring less than or equal to 1.0 cm in its greatest dimensionth edition of the American Joint committee on Cancer (AJCC) tumour, node, metastasis (TNM) classification system for differentiated thyroid cancer defines T1a tumours as those with a tumour diameter ≤1 cm (PTMC) without extrathyroidal extension (ETE), and this subgroup of patients are recommended to undergo lobectomy. However, the AJCC classification system, along with the guidelines recommended by the American Thyroid Association, defines the tumour size according to the traditional intraglandular maximal tumour diameter, and whether the subgroup of patients with multifocal PTMC and a total tumour diameter (TTD) >1 cm shares the same features and prognosis as those with traditional PTMCs remain unclear.The 6Hence, in the present study, we aimed to demonstrate whether the TTD should be used as a more accurate criterion for tumour size of papillary thyroid carcinomas (PTCs) and should be added as an additional prognostic factor in the AJCC classification system.Overall, the postoperative follow-up period ranged between 18–148 months, with a median follow-up of 61.0 months. Clinicopathological characteristics of the papillary thyroid microcarcinoma patients n = 1102) and papillary thyroid carcinoma  (n = 210) were present in Table  and papivs. a multifocal TTD >1 cm (Group E), the proportions of cases with LNM significantly differed, at 21.6% and 50.3%, respectively (p < 0.001). In addition, comparison was also carried out between cases with a multifocal TTD ≤ 1 cm (Group D) vs. multifocal TTD >1 cm (Group E), which revealed significant differences in LNM   and in 34.1% of PTMCs with a multifocal TTD >1 cm and ≤2 cm (Group B) (p = 0.522). For PTMCs, LNM occurred in 21.6% and 25.1% of patients with a unifocal diameter ≤1 cm (Group C) and a multifocal TTD ≤ 1 cm (Group D), respectively (p = 0.286). However, when comparing PTMCs with a unifocal diameter ≤1 cm (Group C) vs. multifocal TTD >1 cm (Group E)   and in 50.9% of PTMCs with a multifocal TTD of >1 cm and ≤2 cm (Group B) p = 0.838). For PTMCs, ETE occurred in 25.6% of patients with a unifocal diameter ≤1 cm (Group C) and 19.4% of patients with a multifocal TTD ≤ 1 cm (Group D) (p = 0.068). However, when comparing PTMCs with a unifocal diameter ≤1 cm (Group C) to those with a multifocal TTD >1 cm (Group E), the ETE proportions significantly differed, at 25.6% and 35.8%, respectively (p = 0.005). In addition, significant differences were also observed between cases of multifocal TTD ≤ 1 cm (Group D) 8. For PTvs. a multifocal TTD >1 cm (Group E), the proportions of cases with local infiltration were 3.8% and 7.3%, respectively, which showed a statistically significant difference (p = 0.008). In addition, comparison was also carried out between cases of multifocal TTD ≤ 1 cm (Group D) vs. multifocal TTD >1 cm (Group E), although no significant differences were found   and in 11.8% of PTMCs with a multifocal TTD of ≫1 cm and ≤2 cm (Group B) p = 0.439). For PTMCs, local infiltration occurred in 3.4% and 3.3% of patients with a unifocal diameter ≤1 cm (Group C) and a multifocal TTD ≤ 1 cm (Group D) (p = 0.970). However, when comparing PTMCs with a unifocal diameter ≤1 cm (Group C) 39. For PFinally, we examined the relationships between tumour size and the recurrence-free survival (RFS) of the patients. Kaplan-Meier analysis revealed that there was no difference in the RFS between patients with PTCs with a unifocal tumour diameter of ≫1 cm and ≤2 cm (Group A) compared to those with PTMCs with a multifocal TTD of ≫1 cm and ≤2 cm (Group B) (p = 0.788) Fig. . In addi6. PTMC is being diagnosed with increasing frequency worldwide and now constitutes nearly half of all cases of PTC8. The primary operative interventions for PTCs with tumour size >1 cm and PTMC differ6, and, because of this, clinicians are faced with the challenge of detecting and managing multifocal PTMC efficiently.PTC is the most common malignant neoplasm originating from the thyroid gland and generally has an excellent prognosis6. For PTC, including PTMC, the current AJCC TNM staging system defines the tumour size as the greatest dimension of the largest solitary tumour in cases of multifocal tumours6. Multifocality has been reported to occur in 30–40% of PTMCs11. A close relationship between larger tumour size and worse outcome, both in terms of recurrence and survival, has been well established and is generally accepted14. However, it remains unclear whether multifocal PTMCs with a TTD >1 cm are associated with worse outcomes compared to those with a TTD ≤ 1 cm.The TNM staging system is based on the extent of the primary tumour (T), the presence of regional lymph node metastasis (N), and the identification of distant metastasis (M)et al.15 concluded that PTCs >1 cm were associated with a worse prognosis, and more extensive thyroid surgery was thus required for these patients. This finding impacted the decision to revise the AJCC classification system to subdivide the T1 category for intraglandular tumours ≤2 cm into tumours ≤1 cm and >1 cm and ≤2 cm in size. Similarly, the various other systems classifying tumours into high- and low-risk groups include tumour size as a one of the prognostic grouping factors; however, the size thresholds range widely17.A large Surveillance, Epidemiology, and End Results database study of 52,173 patients by Bilimoria In our results, the risks of LNM, ETE, and local infiltration, as well as the RFS, did not significantly differ between PTCs with a unifocal diameter >1 cm and ≤2 cm and PTMCs with a multifocal TTD >1 cm and ≤2 cm or between PTMCs with a unifocal diameter ≤1 cm and multifocal TTD ≤ 1 cm. However, the risks of LNM, ETE, and infiltration and the RFS were significantly different between PTMCs with a unifocal diameter ≤1 cm and multifocal TTD >1 cm and between multifocal PTMCs with a TTD of ≤1 cm and >1 cm, except for local infiltration. The latter finding may be due to the included PTMCs mostly being early-stage tumours and rarely presenting local infiltration.21. Multifocality has been reported to be a negative prognostic factor for recurrence and is considered an important risk factor in many publications26. Further, it is still unclear whether PTMC should be considered a separate clinical entity that requires different management compared to conventional PTC9; therefore, the size of all foci should be considered when estimating the tumour mass for multifocal PTMC.The main argument for treatment such as prophylactic central lymph node metastasis or radioiodine ablation in PTC and PTMC patients is based on maximum clearance of the tumour, which could result in a decreased rate of recurrence, improved survival, and avoidance of reoperations in the central neck compartment1. In this study, we further demonstrated that multifocal PTMCs with a TTD >1 cm were also associated with similar risks of high-risk clinicopathological features  and similar RFS as PTC. Overall, the above findings indicate that, in multifocal microcarcinomas, the diameter of the largest lesion cannot be directly used as an efficient tool to predict the risks of LNM, ETE, and local infiltration, as well as the prognosis.In our previous study, we found that multifocal PTMCs with a TTD >1 cm were associated with a similar risk of LNM as PTCsOur data have some limitations. First, this study was not a randomised case-control trial; however, we consider our findings to reflect the total PTMC patient population surgically treated at one institutions. In addition, our cohort of patients had a relatively short median follow-up of 61 months. A longer follow-up is required to make any definitive conclusions regarding the effect of the TTD of multifocal PTMCs on the RFS.The TTD may represent a more accurate criterion for tumour size of PTMCs. Our results suggest that the classification of tumour size according to the dimension of the largest focus for multifocal PTMCs, as advocated by the AJCC and currently used as a treatment recommendation by the American Thyroid Association, needs to be revisited.We conducted a retrospective review of consecutive patients who underwent total-thyroidectomy plus prophylactic central lymph node dissection in our medical centre  between January 2003 and November 2014. Data were obtained from our clinical database, all methods were performed in accordance with the relevant guidelines and regulations, the study protocol was approved by our institutional review boards  and the file number: 20131001PTMC. Written informed consent from all participants were obtained.A total of 1102 patients with PTMC were included in the analysis. Additionally, 210 patients with unifocal PTC with a diameter of more than 1 cm but less than or equal to 2 cm were also included for comparisons with the PTMC cases. Demographic information, surgical details, histopathologic details, and postoperative use of radioactive iodine were recorded. Postoperative thyroid-stimulating hormone suppression was practiced based on the recurrence risk for all patients. The clinicopathological parameters, including radioactive iodine remnant ablation status were also recorded and compared. The subgroups were organized: Group A: PTC, unifocal with diameter >1 cm and ≤2 cm (n = 210); Group B: PTMC multifocal with total tumor diameter (TTD) >1 cm and ≤2 cm (n = 85); Group C: PTMC unifocal with diameter ≤1 cm (n = 712); Group D: PTMC multifocal with TTD ≤ 1 cm (n = 211); Group E: PTMC multifocal with TTD >1 cm (n = 179).During the past few years in our centre, multifocality was often detected in the postsurgical pathology, and local recurrence was commonly found among PTMC patients who had undergone partial thyroidectomy. For these reasons, since 2003, we perform total thyroidectomy plus bilateral central neck dissection for all PTMC patients, regardless of the size, number of foci, and disease stage. When preoperative imaging tests such as ultrasound indicate suspected LNM in the lateral neck, lateral neck dissection is performed. Fine-needle aspiration biopsy and/or intraoperative frozen section examination is routinely performed during thyroid surgical procedures. False-negative cases who underwent lobectomy and in whom more than one microcarcinoma focus was found by routine pathology undergo residual thyroid resection with central lymph node dissection; that is, resection of the thyroid gland and the adjacent nodes bordered superiorly by the hyoid bone, inferiorly by the brachiocephalic vessels, and laterally on each side by the carotid sheaths.Routine pathological examination was performed on the whole specimen with serial sectioning at 3-µm intervals for hematoxylin and eosin staining, and the tumours were diagnosed by two experienced pathologists from the Department of Pathology of Union hospital according to the criteria of the World Health Organization. Multifocality was considered when two or more foci were found in one or both lobes; for multifocal PTMC, the largest, dominant tumour was first analysed. Subsequently, the TTD was calculated as the sum of the maximal diameter of each lesion and used for further analysis. Recurrence/disease-persistence was defined as the appearance of pathologically proven malignant tissue and/or the appearance of metastatic lesions in the lungs, bones, and/or brain by imaging studies.The patient parameters were compared using Student’s t-test for continuous variables and the Chi-square or Fisher’s exact test for categorical variables. Continuous variables are presented as the mean ± standard deviation or as medians and ranges, and categorical variables are presented as percentages and absolute numbers. Multivariate analysis of all variables was performed using a logistic regression model. Survival outcomes were analysed using the Kaplan-Meier method and log-rank test. All reported p values in this study are 2-sided, and p values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS software, version 13.0 .


To examine whether the relationship between the metabolic syndrome (MetS) and various sleep parameters  varies by age.N = 19,691) of middle-aged (aged 45–64 years) and older (aged ≥65 years) subjects. Habitual sleep duration , sleep disturbances , and symptoms of sleep-disordered breathing  were measured by questionnaires.Waist circumference, blood pressure, triglycerides, high-density lipoprotein cholesterol, and fasting glucose were used to determine MetS status in a cohort (P < 0.01 for sleep duration × age). In the entire cohort, having at least one SDB symptom ≥4 times per week was linked to an increased prevalence of MetS; however, the PR was higher in middle-aged subjects compared with older subjects . Finally, independent of subjects’ age, reports of sleep disturbances  were associated with a higher likelihood of having MetS .Among the participants, 4,941 subjects (25.1%) fulfilled the criteria for MetS. In the entire sample, both short and long sleep durations were associated with higher prevalence of MetS as compared to normal sleep duration. When stratified by age, a similar pattern was observed for middle-aged subjects  [95% CI], 1.13 [1.06–1.22] for short sleep and 1.26 [1.06–1.50] for long sleep duration). In contrast, in older individuals (≥65 years old), only long sleep duration was linked to a higher prevalence of MetS (1.26 [1.12–1.42]; Our results suggest that age may modify the associations between some sleep parameters and the prevalence of MetS. The metabolic syndrome (MetS) is defined as a cluster of several cardio-metabolic risk factors. This includes hypertension, hyperlipidemia, hyperglycemia, reduced blood concentrations of high-density lipoprotein (HDL) cholesterol, and abdominal obesity . Accumuln = 32,929). In contrast, long sleep duration (≥ 8 h) was associated with lower odds of having prediabetes in adults aged <40 years , short sleep duration (≤5 h) was associated with higher odds for prediabetes. Long sleep duration was, however, unrelated to prediabetes in older subjects  in which self-reported sleep duration was not associated with hypertension among those aged 60 years and above  and prediabetes—defined by the authors as blood levels of glycated hemoglobin ≥5.7% —was found in Japanese subjects aged <40 years , sleep disturbances , and SDB symptoms (such as snoring and sleep apneas) with MetS vary by age . We hypothesized that poor sleep patterns increase the prevalence ratio (PR) of MetS.www.epihealth.se). At the time of retrieving data for the present analysis, the EpiHealth study was still recruiting subjects. This explains the difference in initial sample sizes between previous publications , two participants were excluded because their age was more than two SD apart from the population mean; 360 individuals were then excluded because of missing data on biochemical parameters or waist circumference. Finally, 437 participants had missing data on covariates, and 44 subjects provided no reports on sleep duration. After exclusions, data from 19,691 subjects  were available to investigate the association between sleep duration and MetS. From them, 19,142  participants had complete data on sleep disturbance and 16,467  on SDB symptoms.From the initial sample size . Blood pressure was recorded twice in the sitting position by trained personnel with automatic device . Waist circumference was measured at the umbilical level. Blood was collected for determination of fasting glucose, LDL- and HDL-cholesterol, and serum triglycerides at the hospital laboratory using an Architect Ci8200 analyzer  . The samMetabolic syndrome was defined as the presence of at least three of the following conditions: elevated waist circumference ; hypertriglyceridemia, defined as a serum triglyceride concentration ≥150 mg/dL [≥1.7 mmol/L]; low HDL cholesterol (<40 mg/dL [<1.0 mmol/L] for men and <50 mg/dL [<1.3 mmol/L] for women); elevated blood pressure (systolic ≥130 and/or ≥85 diastolic mmHg) or antihypertensive drug treatment .Participants were asked to indicate how many hour per day they usually sleep . The answer “don’t know/don’t want to answer” was treated as missing value. Short sleep duration was defined as sleep ≤6 h per day, normal sleep duration corresponded to 7–8 h sleep per day, and sleep ≥9 h per day was defined as long sleep duration.Sleep disturbances were determined based on the following symptoms: difficulties in falling asleep, early awakenings, difficulties getting back to sleep after nighttime awakenings, and disturbed sleep. Symptoms of SDB included witnessed sleep apnea and heavy snoring (witnessed or according to participate him/herself). Participants were required to indicate the frequency of each symptom by the following options: “never/seldom,” “1 to 3 times a month,” “1 to 3 times a week,” “4 or more times a week,” and “ don’t know/don’t want to answer.” Participants who reported that they suffered from at least one of the above-mentioned sleep disturbance symptoms for “4 or more times a week” were defined to have a sleep disturbance. Participants who indicated to experience either witnessed sleep apnea or heavy snoring for “4 or more times a week” (or both) was defined to have SDB symptoms. The option “don’t know/don’t want to answer” was treated as missing value.Age and gender were recorded in the test center. Participants’ educational attainment, physical activity (PA) during leisure time, alcohol consumption frequency, and current smoking were assessed by the Internet-based questionnaire. Participants’ educational attainment was defined as primary and elementary school , upper secondary school , university, or other . PA during leisure time was measured on an eight-point scale. A low level of PA was defined as spending most leisure time mostly sedentary or having light PA about 2–4 h per week, such as walking, gardening, and light housework, etc. A medium level of PA was defined as moderate PA at least 1–2 times a week, such as jogging, swimming, heavy gardening, etc., or light PA for more than 4 h per week or taking care of all the housework, both light and heavier. A high PA level was defined as more strenuous PA at least three times a week, such as playing tennis, swimming, and running, etc. Based on responses to six questions about smoking habits, the participants were assigned to non-smokers or smokers. Alcohol consumption frequency during the last 12 months was categorized as “never,” “≤1 time/week,” “2–3 times/week,” and “≥4 times/week,” yielding a four-level ordinal variable.U test.All statistical analyses were performed using SPSS version 22.0 . Descriptive data are presented as mean (SD) for continuous variables and as percentages for categorical variables. The Pearson chi-square test was used to analyze group differences for categorical variables. Numerical data were analyzed with Mann–Whitney Binomial regression with log link function was performed to examine associations between the prevalence of MetS and sleep parameters. This statistical approach was used because the outcome (MetS) was common (25%) in the present cohort. Under such circumstances, log-binomial regression analysis is generally considered to be more conservative than logistic regression . PRs dern = 9,061, 44%) and risk of potential misclassification. Additionally, shift work was the only variable that was not significantly associated with MetS in fully adjusted models. Finally, comparison of regression models using Akaike’s Information Criterion (AIC) revealed that models not adjusted for shift work had lower AIC, indicating a better model fit.Note that job-related questions (including those on shift work history) were presented only to participants who were still working at the time of the online survey . Hence, P > 0.05). Possible multiplicative interaction effects of sleep parameters with age (in y) on MetS risk were investigated in fully adjusted models . We considered interaction present at P < 0.05.Potential confounders were selected based on existing information on risk factors for impaired sleep and MetS using the method of directed acyclic graphs (DAGs) . DAGs isThe proportion of missing data on main exposure variables was less than 1% for sleep duration, 3% for sleep disturbance, 17% for SDB symptoms, and ≤1% for covariates. To assess if exclusions of subjects because of missing values could have affected the observed associations between sleep variables and the prevalence of MetS, we performed multiple imputation with the assumption that data were missing at random. The imputation procedure resulted in five imputation data sets.Of the total 19,691 participants with complete data on sleep duration, 21% of middle-aged subjects (age <65 years) and 31.2% of older individuals (≥65 years) met criteria for MetS, respectively. Compared with middle-aged subjects, older individuals had more often an elevated waist circumference , hypertension (systolic ≥130 and/or ≥85 diastolic mmHg or antihypertensive drug treatment), elevated serum triglycerides (≥150 mg/dL [≥1.7 mmol/L]), and elevated fasting glucose (≥110 mg/dL [≥6.1 mmol/L] or drug treatment for diabetes). In contrast, low HDL cholesterol level (<40 mg/dL [<1.0 mmol/L] for men and <50 mg/dL [<1.3 mmol/L] for women) did not differ between age groups. For more details, see Table P = 0.009). Similar results were obtained for the interaction between sleep duration and age when the latter was treated as binary variable (P = 0.028). Compared to normal sleep duration, long sleep duration increased the prevalence of MetS in both middle-aged and old participants . In contrast, short sleep duration was linked to a higher prevalence of MetS in middle-aged but not older subjects .Results are summarized in Table A secondary analysis was performed dividing the sleep duration variable into four instead of three categories . In the entire sample, for those who reported to sleep ≤5 or ≥9 h per day a higher prevalence of MetS was found , compared with those who slept between 7 and 8 h per day. No such difference in the prevalence of MetS was observed between the 6 h-sleep duration and reference groups (1.05 [0.99–1.10]). When stratified by age, a similar pattern was observed among middle-aged subjects . In contrast, in the ≥65 years age group only long sleep duration was linked to a higher prevalence of MetS (1.26 [CI 1.12–1.42]).P = 0.189 for sleep disturbance × age (in years); P = 0.521 for sleep disturbance × age group]. Participants with sleep disturbance were more likely to have MetS, than those without sleep disturbance . A separate analysis in the entire cohort, as well as within the age groups, revealed that the higher the number of SDB symptoms, the higher the prevalence of MetS  was performed. This analysis demonstrated that early and older midlife age groups had a higher prevalence of MetS when reporting short sleep duration (≤6 h per day). In contrast, long sleep duration (≥9 h per day) was linked to a higher prevalence of MetS only in the older midlife age group and among individuals ≥65 years old . Finally, in all age groups an association between SDB symptoms and increased prevalence of MetS was noticed, with highest PR for subjects of the early midlife age group .Our study demonstrates that the association between sleep duration and MetS varies by age. In middle-aged participants (45–65 years), both short (defined as ≤6 h sleep per day) and long (defined as ≥9 h sleep per day) duration sleepers exhibited an increased prevalence of MetS, compared with normal-duration sleepers (defined as 7–8 h sleep per day). In contrast, in older individuals (aged ≥65 years), long but not short sleep duration was linked to a higher likelihood of having MetS. Collectively, our study provides an important piece of evidence that the relation between sleep duration and metabolic health may change during adulthood. Age-specific associations between sleep duration and MetS components have also been observed by others. For instance, a study using the NHANES I follow-up data showed that daily sleep durations of ≤5 h compared to 7–8 h were associated with a significantly increased risk of incident hypertension in participants aged 32–59 years. This association was, however, not found among people aged ≥60 years . In a crSleep-disordered breathing comprises alterations in respiratory rate, rhythm, and depth present during sleep . Obstrucn = 4,197) showed that problems with falling and staying asleep were associated with an increased prevalence of MetS   . Specifictively) . Howeverctively) .A major strength of this study is that the analysis was based on a relatively large sample. Moreover, results were robust to adjustments for multiple potential confounders, such as lifestyle factors. To our best knowledge, our study is also among the first to investigate systematically how various characteristics of poor sleep link to the risk of MetS in middle-aged and older subjects. Several limitations, however, apply to our cross-sectional study. It cannot prove cause and effect. Another limitation is that sleep variables were based on self-reports. Thus, our observations should be confirmed by further studies utilizing objectively measured sleep parameters. An additional limitation of our study is that no measures of circadian misalignment have been collected. Sleeping 7–8 h during circadian improper time windows  has been shown to adversely affect metabolic health , 34. MorIn our study, we demonstrate that both sleep duration outside 7-8 hours per day and having at least one SDB symptom ≥4 times per week increase the risk of MetS in an age-specific manner. Sleep disturbances  were also associated with an increased prevalence of MetS. The latter relationship was not modified by subject’s age. Given the high prevalence of sleep problems and metabolic perturbations in modern society, educational programs aiming to optimize sleep could, therefore, represent promising interventions to improve metabolic health in middle-aged and older subjects.The Ethics Committee at Uppsala University approved the general procedures of the EpiHealth study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. An additional ethical approval for the current data analyses was obtained from the Ethics Committee at Uppsala University. A short description of the research proposal was displayed on EpiHealth’s homepage for 1 month which allowed participants in EpiHealth study to withdraw their consent.OT: wrote the manuscript, performed the literature search, data and statistical analysis, and data interpretation. EL: reviewed and revised the manuscript, and supported data interpretation. SE: contributed to the study design, acquired data, and gave scientific advice. LL: contributed to the study design, acquired data, and reviewed the manuscript. HS: reviewed and revised the manuscript. CB: reviewed and revised the manuscript, supported data interpretation, and gave scientific advice. All authors approved the final version of the manuscript. OT and CB had full access to all of the data and take responsibility for the integrity and accuracy of the data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GC) is a multistep and multistage disease and the majority of GC cells could overexpressed one or more oncogenes. Trop2 and amphiregulin (AREG) are both overexpressed in various epithelial cell cancers and have the role in the increases tumor cells division and metastasis. However, little is known about the function and correlation of two oncogenes coexpressed in GC. The expression level of these two genes in 791 cases of GC tissues were tested, the correlations between two genes expression and clinical pathological characteristics and overall survival in GC patients through immunohistochemistry (IHC) were analyzed. This study also explored the mRNA expression level of two genes in 26 cases of freshly GC tissues by qRT‐PCR. The results indicated that Trop2+/AREG+ coexpression was higher in GC tissues than in adjacent tissues. Trop2+/AREG+ protein coexpression were associated with Tumor Node Metastasis (TNM) stage , tumor size , lymph node metastases , and distant metastases . GC patients with Trop2+ and AREG+ protein coexpression had poor overall survival rates . The expression level of Trop2/AREG were positively correlated (r 0.254 and P < 0.001). The result of the mRNA expression was similar to that of the protein expression. Overall, Trop2 and AREG could be seen as prognostic cobiomarker in GC and combined detection of Trop2 and AREG could be viewed as helpful in predicting the prognosis of the GC patients.Gastric cancer ( Gastric cancer (GC) is one of the five common types of tumors and ranks the third leading cause of cancer‐related mortality worldwide The human trophoblast cell surface glycoprotein (TACSTD2/Trop2/M1S1/GA733‐1) was first identified in the surface of the human trophblast cells and located at chromosome 1q32 AREG is a member of epidermal growth factor (EGF) family and has a critical role in growth control, although the precise role has not been understood deeply. AREG is located at chromosome 4q13.3 and shedding ECD of original a 252 amino acid transmembrane precursor (pre‐AREG), then secret into blood or other cell surrounding microenvironment through autocrine and paracrine. Emerging evidence showed that AREG may be important for tumor metastasis and resistance to therapy Our lab have been studied two papers about the expression of two genes in gastric cancer, respectively A total of 791 formalin‐fixed, paraffin‐embedded (FFPE) stomach tissue samples were collected from 741 patients, including 589 cancer, 89 matched tumor neighbor, 65 chronic gastritis, 26 intestinal metaplasia, and 22 intraepithelial neoplasia. All FFPE specimens were obtained from the Department of Pathology, Affiliated Hospital of Nantong University from 2003 to 2010. The associated clinical records of the donor patients including age, sex, tumor node metastasis (TNM) stage, histological type, differentiation status. No patients received any types of treatments  before surgery. Overall survival (OS) was defined as the period from initial biopsy confined diagnosis to death. Information on patients who were alive at the last follow‐up date was deleted from the analysis and follow‐up from 2 to 10 years. Additional 26 freshly gastric cancer and matched tumor neighbor tissues were obtained primarily from the First Affiliated Hospital of Nanjing Medical University, Huai'an Second People's Hospital, and Affiliated Hospital of Nantong University. The study protocol was approved by the Human Research Ethics Committees of these hospitals. All patients provided written informed consent for their stomach tissue samples to be used for research.™ RT reagent kit  in accordance with the manufacturer's instructions. Human β‐actin served as the internal control for determining Trop2 and AREG mRNA levels. The primers as follows: human β‐actin forward, 5′‐ TGGAGAAAATCTGGCACCAC‐3′, and reverse, 5′‐GATGATGCCTCGTTCTAC‐3′, and Trop2 forward, 5′‐TGTCCTGATGTGATATGTCTGAG‐3′, and reverse, 5′‐GGGTGAGAGTGGGTTGGG‐3′, AREG primer: forward, 5′‐GCTGTCGCTCTTGATACTCG‐3′, and reverse,5′‐.Total RNA was extracted from freshly samples using TRIzol reagent  and reverse transcribed into cDNA using a PrimeScript−∆∆Ct Method.ACGCTTCCCAGAGTAGGTGT‐3′ . Comparative quantification was determined using the 2χ2 test were performed to determine the statistical significance of differences groups. The X‐tile software program  vs. “high”) using predeterminate cut‐off values P‐value of less than 0.05 was considered as statistically significant.The data for statistical analyses were performed by using the SPSS 18.0 statistical software package . The Student's t‐test and Pearson http://www.tissuearray.org/rimmlab), we identified the cut‐off point according to overall survival in GC tissues. For Trop2 and AREG, the cutoff 130 was selected:0–130 was considered “low or no” expression, whereas 131–300 was considered “high” expression. High Trop2 and high AREG (T+A+), high Trop2 and low AREG (T+A‐), low Trop2 and high AREG (T‐A+), low Trop2 and low AREG (T‐A‐) expression were detected, respectively, in gastric tissues, which including cancer, matched adjacent tissue, chronic gastritis, intestinal metaplasia, intraepithelial neoplasia , tumor size, lymph node metastases , and distant metastases. However, no correlation was found between Trop2 and AREG expression levels and gender, age, histological type, differentiation . Both Trop2+ and AREG+ group coexpression level was the highest (41.42%) than T+A‐(24.62%), T‐A+(21.39%) and T‐A‐(12.56%) groups in cancer tissues and the T+A+ expression level in cancer tissues were higher than that in the matched adjacent tissue. The results also indicated that high Trop2 and AREG protein coexpression was associated with TNM stage, lymph node metastases and distant metastases. Furthermore, high level of Trop2 and AREG expression (T+A+) predicted poor overall survival. The expression level of two genes was positively correlative in GC tissues, and the similar results were found with regard to the mRNA expression level between two genes.It is well‐known that GC was developed from normal gastric mucosa to chronic gastritis, chronic atronic gastritis, intestinal metaplasia, atypical hyperplasia  to cancer. The turning point of malignancy changes is the status of intestinal metaplasia The overexpression of oncogene Trop2 in several types of solid tumor cancers had been provided AREG, as a ligand for the EGFR, is a transmemebrane tyrosine kinase receptor, which plays a central role in regulating cell division and death This study has several limitations: first, this study is a retrospective observation examine and the gastric tissues of patients were obtained from 2003 to 2010, the conclusion might not be suitable for the current population; second, IHC used in this study is a semiquantitative method, additional study in vivo and in vitro should be done in the future to confirm the role of Trop2 and AREG in cancer cells; Third, the number of genes coexamined in this study were relatively small and other useful candidate genes should be studied.In conclusion, high Trop2 and AREG expression is associated with poor prognosis and may be considered as an independent prognostic cobiomarker in GC.The authors have declared that no competing financial interests exist.


The aims of the study were to A retrospective cross-sectional study using data from the Swedish Pregnancy Register 2011 to 2013  was performed. Additionally, guidelines on prenatal diagnosis were collected. Biostatistical and epidemiological analyses were performed including calculation of odds ratios (OR) and their 95% confidence intervals in univariate and multivariate logistic regression analyses.The national uptake of routine ultrasound examination, Combined Ultrasound and Biochemical test (CUB), Amniocentesis (AC) and Chorionic Villus Sampling (CVS) were 97.6, 33.0, 2.6 and 1.1%, respectively. From 2012, 6/21 counties offered CUB test to all pregnant women, nine counties at specific indications, and five counties did not offer CUB at all. Advanced maternal age demonstrated the highest impact on uptake of prenatal diagnosis. Further, university educational level in relation to lower educational level was associated with an increased likelihood of undergoing CUB , AC  and CVS .Offers of prenatal diagnosis varied considerably between counties resulting in unequal access to prenatal diagnosis for pregnant women. The intentions of the Swedish Health and Medical Services Act stating equal care for all, was thus not fulfilled. Antenatal care (ANC) is free of charge which almost all pregnant women in Sweden attend . Most prThe Swedish Maternal Health Care Register (MHCR) is a national quality register where pregnant women participate on a voluntary basis by contributing with information on their pregnancy and delivery . MHCR haThe National Board of Health and Welfare in Sweden has issued regulations concerning counselling on different methods of early prenatal screening and diagnostic procedures . These rThe rationale of this study was to investigate the utilisation of prenatal diagnosis in Sweden during the study period 2011 to 2013 in relation to the different offers of prenatal screening and diagnostic procedures on a national level and comparing different counties.The overall aim was to investigate background characteristics and pregnancy outcomes in relation to the use of prenatal screening methods and diagnostic procedures in Sweden.i) investigate guidelines on prenatal diagnosis in the counties of Sweden, ii) investigate uptake of routine ultrasound examination, combined ultrasound and biochemical test (CUB), chorionic villus sampling (CVS) and amniocentesis (AC), and iii) background characteristics and pregnancy outcomes in relation to different prenatal screening and diagnostic procedures.The specific aims of the study were to N = 43) in Sweden for 2011, 2012 and 2013. The guidelines during this study period were almost consistent with the exception of changes in two counties where no pregnant women previously had been offered CUB until 2011. A new guideline was introduced during 2012 in these two counties, offering all pregnant women CUB. The proportion of births in these two counties corresponds to 4% of all births in Sweden. Sweden includes 21 different counties where the majority of counties host only one MHCA, whereas some larger cities host multiple MHCAs, as for example the area of the capital Stockholm. Results related to guidelines will be presented on county-level.This retrospective, cross-sectional, epidemiological study analysed data on pregnancies from the Swedish Maternal Health Care Register and the Swedish Pregnancy Register from 2011 to 2013. MHCR was an independent register until 2012. MHCR was integrated into the SPR, as one of three registers when the SPR was formed in 2013. Here, SPR refers to MHCR and SPR as one entity. Inclusion criteria, for participating in the study, were being a subject included in the SPR with a date of delivery of a live or stillborn child from January 1st 2011 to December 31st 2013, and with a gestational age of 22 weeks and 0 days to 43 weeks and 0 days. Data on all pregnancies 2011 to 2013 were obtained from the SPR, comprising 284,789 women and their offspring. The participation rate of pregnant women in SPR was during 2011, 2012, and 2013, 81, 85 and 89%, respectively. The coverage of variables in relation to county, varied from 74 to 99% during 2013. Additionally, medical guidelines regarding offers to pregnant women on prenatal screening and diagnostic procedures were collected from each Maternal Health Care Area . Primiparity was defined as having delivered one child, i.e. including the index pregnancy, and multiparity was defined as having delivered at least 2 children . Body mass index (BMI) was calculated with the formula BMI = kg/m2. The different BMI groups were defined in accordance with the WHO’s definition of BMI: underweight: <18.50 kg/m2; normal weight: 18.50–24.99 kg/m2; overweight: 25.00–29.99 kg/m2, obesity class 1: 30.00–34.99 kg/m2, obesity class 2: 35.00–39.99 kg/m2, and obesity class 3: ≥40.00 kg/m2 [Level of education was defined as elementary school, high school or university. Employment status was categorized into “employed”, “student”, “parental leave”, “unemployed”, “sick leave”, and “other status”. Country of origin was categorized into Sweden, other Nordic countries  and Europe (excluding Sweden and other Nordic countries), Africa, Asia and other countries. The variable Alcohol screening (Alcohol Use Disorder Identification Test = AUDIT scores) was categorized in whether performed “yes” or “no”. AUDIT-score ranged from 0 to 40 scores, and was categorized into ≤5 scores and ≥6 scores, which indicates harmful alcohol use [Self-rated health prior to pregnancy was categorized in “very good”, “good”, “neither good nor poor”, “poor”, and “very poor”. The following variables were categorized in “yes” or “no”: smoking at 3 months prior to pregnancy, smoking at first ANC visit, smoking at 32 weeks of gestation, use of snuff 3 months prior to pregnancy, use of snuff 3 months at first ANC, use of snuff at 32 weeks of gestation, counselling due to fear of childbirth where fear of childbirth was defined in the SPR as a subject being referred for counselling due to fear of childbirth, treatment of psychiatric disorder where psychiatric disorder was defined in the SPR as either medical or psychological treatment of psychiatric disorder, or both, combined ultrasound and biochemical test (CUB), chorionic villus sampling (CVS) and amniocentesis (AC).00 kg/m2 . Level oohol use . Self-racombined ultrasound and biochemical test (CUB), chorionic villus sampling (CVS), amniocentesis (AC), counselling due to fear of childbirth and treatment of psychiatric disorder. Gestational age was reported in days of gestation and presented as a continuous variable. Mode of delivery was categorized in “vaginal delivery”, “instrumental delivery” (including delivery with vacuum extraction or forceps), and “caesarean section”. Caesarean section (CS) was further categorized in “elective caesarean section” and “emergency caesarean section”. Birth weight in grams was presented as a continuous variable.The following variables were categorized into “yes” or “no”: p < 0.05. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated in univariate and multivariable logistic regression analyses. SPSS vs. 22 and vs. 23 were used for these calculations. A Venn diagram was created to present the uptake of CUB, CVS and AC in the study sample. A figure presenting a map of Sweden was created to illustrate geographical differences in uptake of CUB, where the 21 counties were categorized into 4 groups of CUB uptake rate: less than 10%, 10 to 29.99%, 30 to 69.99% or 70% or more.Categorical variables were analysed with frequencies and percentages. Continuous variables were presented by their mean value and standard deviation (SD), and by their median value and interquartile range (IQR). Continuous variables were tested for the assumption of normal distribution. Test of trend was analysed by Linear-by-Linear Association for investigation of linear trends over the years. Test of difference between independent groups were analysed with One-Way Anova test and independent samples t-test for parametric data, corrected for homogeneity for variance if necessary. The Pearson’s Chi-Square test was used for test of difference between groups for categorical data. Level of significance was set at All counties in Sweden except one had issued written guidelines concerning offers of prenatal screening methods and diagnostic procedures during the first and second trimesters of pregnancy. These guidelines remained unchanged in all counties except for two counties, during the study period. Three Swedish counties offered a routine ultrasound examination at 12 weeks of gestation for the purpose of dating and all other counties offered a second trimester scan at the gestational age of 17–20 weeks, with the exception of one county that accepted dating from week 16. CUB was offered to all pregnant women in six counties, was offered on indication advanced maternal age in nine counties, and was not offered at all in five counties. The definition of advanced maternal age as indication for CUB demonstrated a substantial variation between counties. The different cut-off-values defining an indication for offering the CUB test were: age >33 years at last menstrual period, age ≥35 years at last menstrual period, age ≥35 years at conception, age ≥35 years at the time for the CUB-test, and age ≥35 years at the estimated date of delivery. In addition, one county included “anxiety related to pregnancy” as an indication for offering CUB. All counties offered either CVS or AC as prenatal diagnostic procedures on the indications: maternal age ≥35 years, increased risk for chromosomal aberration following CUB, or second trimester serum screening, or familial genetic condition. Three counties offered AC only if CUB had previously been performed and indicated an increased risk. During 2013, the uptake of CUB varied between the counties from 2.2% to 80.3%,  (Table n = 1252) thereafter underwent CVS, and 2.7% (n = 2493) underwent AC after CUB. Of all women who were examined with CVS (n = 2970), 42.2% had undergone CUB prior to CVS. Of all women who were examined with AC (n = 7279), 34.2% had undergone CUB prior to AC.The study population included 284,789 pregnant women, and the distribution of participants per year was 30.9% (2011), 33.7% (2012) and 35.3% (2013). Background characteristics for the study population are presented in Table p-value <0.001) .Pregnant women who underwent CUB, CVS or AC reported being employed in significantly higher proportions , vs. 65% for women who had not undergone CUB, CVS or AC   Table .p-value <0.001) at all three check points, i.e. 3 months prior to pregnancy, at first antenatal visit and at gestational age of 32 weeks, by women who had undergone CUB compared to those who had not been examined by CUB, CVS or AC, i.e. “all others”   Table . There w1) Table .p-value <0.001)  and 60.9% (emergency CS), . When the OR was adjusted for educational level the OR still remained increased . Table Maternal age demonstrated the highest impact on the likelihood of undergoing invasive prenatal diagnosis . Further, women who had achieved an educational level corresponding to university had an increased likelihood of undergoing AC  and CVS , in relation to women with a lower educational level. Their increased odds ratios for AC and CVS remained unchanged after adjusting for maternal age . Additionally, pregnant women who had received counselling due to fear of childbirth demonstrated a higher likelihood of undergoing AC or CVS , in comparison to those who had not been counselled for fear of childbirth. When adjusted for age and educational level the likelihood remained significantly increased for AC  and CVS .A decreased likelihood of undergoing AC or CVS was demonstrated for pregnant women who reported country of birth outside of Sweden, compared to those who were born in Sweden . When adjusted for age and educational level the odds ratios remained significantly decreased .The aim of this study was to make a national survey on guidelines concerning offers on prenatal diagnosis in Sweden. Further, we aimed to investigate background characteristics and pregnancy outcomes in relation to the uptake of different prenatal diagnostic methods. During the study period of 2011 to 2013 in Sweden there was an absence of a national consensus regarding guidelines on offers of prenatal diagnosis. The Swedish law states that all pregnant women should be offered information on prenatal diagnosis . HoweverDuring the study period, the SPR did not include data on Non-Invasive Prenatal testing (NIPT). This method is based on analysis of cell-free fetal DNA in maternal blood . In SwedDuring the study period, the Swedish Pregnancy Register demonstrated a satisfactory coverage of pregnancies and moreover, all counties of Sweden are represented in the SPR. Data in the Swedish Medical Birth Register for 2012, demonstrate a mean maternal age, for primiparous women, of 28.4 years, and a mean BMI  of 24.8 , and theOffers of prenatal diagnostic procedures varied considerably between counties in Sweden. Maternal age, as expected, demonstrated the strongest association with the uptake of CUB, AC and CVS. Further, educational level was a strong predictor of uptake of prenatal diagnosis. These circumstances result in an unequal access of prenatal diagnostic tests for pregnant women. The intentions of the Swedish Health and Medical Services Act stating that equal care should be provided for all, was thus not fulfilled. Expecting couples should be offered the same opportunities on prenatal diagnosis nationally.


Pediculus humanus capitis by hot air, soaking in various fluids or asphyxiation using occlusive treatments. In this study, we assessed the maximum time that head lice can survive anoxia (oxygen deprivation) and their ability to survive prolonged water immersion. We also observed the ingress of fluids across louse tracheae and spiracle characteristics contrasting with those described in the literature. We showed that 100% of lice can withstand 8 h of anoxia and 12.2% survived 14 h of anoxia; survival was 48.9% in the untreated control group at 14 h. However, all lice had died following 16 h of anoxia. In contrast, the survival rate of water-immersed lice was significantly higher when compared with non-immersed lice after 6 h , and 24 h . Although water-immersed lice did not close their spiracles, water did not penetrate into the respiratory system. In contrast, immersion in colored dimeticone/cyclomethicone or colored ethanol resulted in penetration through the spiracles and spreading to the entire respiratory system within 30 min, leading to death in 100% of the lice.Chemical, physical, and mechanical methods are used to control human lice. Attempts have been made to eradicate head lice  Pediculus humanus capitis De Geer, 1778) has a global distribution and affects individuals of broad economic or social status worldwide, particularly school-aged children [Acinetobacter spp., Bartonella quintana, and Borrelia recurrentis in head lice [Rickettsia prowazekii [Lice are obligate human hematophagous ectoparasites belonging to the Pediculidae family ,54. Pedichildren ,25. Seveead lice . Furtherowazekii ,41,49 buowazekii ,39. The owazekii ,27,40.in vitro or in clinical trials [Different chemical, physical, and mechanical methods are used to control pediculosis. Use of insecticides such as pyrethrin or malathion was the treatment of choice until recently ,16. Howel trials ,44,48,57l trials ,44, wherl trials ,11.The efficacy of lice infestation treatment has been compared using a variety of protocols, including: hot and cold air ,22, machAccordingly, we conducted the following experiments to investigate: (i) how long head lice can survive under anoxic conditions, (ii) the ability of head lice to survive up to 24 h when immersed in water, (iii) the closure of their spiracles when immersed in water, and (iv) the penetration of water and other products into the respiratory system of head lice.Comité de Protection des Personnes  of the Ethics Committee CPP- Ile-de-France X (2017-02). Informed consent was obtained from all patients.The protocol was reviewed and approved by the Pediculus humanus capitis were obtained by combing the hair of infested patients of 2–68 years of age attending the parasitology department of the Avicenne Hospital, Bobigny, France. Head lice were placed in plastic Petri dishes and were examined for activity and morphological integrity under a binocular magnifier (Zeiss Stemi 2000C). Tests were performed using living, non-injured lice. We started experiments immediately after lice collection. All experiments were done under the same conditions of diet, temperature of water (25 ± 2 °C), and ambient air .From September 2015 to July 2016, TM Compact plastic pouch . Then, the AnaeroGenTM Compact sachet  was quickly placed in the pouch. The system was tied shut with a clip. Each group endured anoxia for one time point . Each test group had a control group (n = 30) that was placed in a plastic Petri dish under ambient conditions . The pouches were then opened and viable and dead lice were counted separately (≤ 1h) for the test and control groups, respectively. Death was defined as the absence of vital signs such as movement of antennae, movements of legs, and gut peristalsis. Each experiment was performed in triplicate.To evaluate the effect of anoxia on head lice survival, ten groups of 30 lice each were used. Each group (n = 30) was placed in a plastic Petri dish (5 cm in diameter) and was placed in an AnaeroGenSeven groups of lice (n = 7–18 each) were placed in plastic Petri dishes (5 cm in diameter) and kept immersed in 10 mL of tap water (24 °C) for H1 to H6, and H24. Petri dishes were gently agitated until all lice were completely submerged and resting at the bottom, and then covered with their lid to prevent water evaporation. After the immersion period, water was removed and live or dead lice were counted. Seven groups of control lice (n = 7–18 each) were placed in Petri dishes without water under room conditions  for the corresponding duration  and were processed similarly. The 7 to 18 lice used in each group were of both sexes. The experiments were performed in triplicate. Death was defined as described above.Three groups of lice (n = 3 each) were placed into a 1.5 mL microfuge tube containing 1 mL of tap water and kept immersed for 10 min, 3 h or 24 h. Control groups were placed into 1.5 mL tubes without water. Upon completion, lice were frozen at −20 °C to preserve the status of the spiracles (SEM1). A similar series of experiments (SEM2) was performed except that −20 °C refrigeration was replaced by liquid nitrogen immersion to avoid rapid movement of the spiracles. After a rapid thaw, specimens were mounted on aluminium stubs on double-sided conductive carbon tabs and positioned on the stubs using a stereoscopic microscope. Samples were analyzed using a FEI Quanta FEG 250 environmental scanning electron microscope operating in LowVac mode, enabling the observation of resin coated samples without metallization and ESEM mode, which is normally used for the observation of wet samples . The chamber pressure was 3 Torr, the Horizontal Field Width was 63.5 μm, and the working distance was 10 mm with a tilt of −1 °. The images were recorded at 1536 x 1103 pixel magnification.®, Cooper, France). The latter contains 4% w/w dimeticone in a volatile silicone base (cyclomethicone 5).Penetration or absence of penetration of liquids through the spiracles into the respiratory system was studied using a water-based stain, an absolute ethanol-based stain, and dimeticone/cyclomethicone-based stain (PouxitThe stain (0.1%) in water and absolute ethanol was brilliant blue FCF (E133) . The stain (1%) in dimeticone/cyclomethicone was a red lipophilic colorant .Nine groups of lice (n = 3 each) were placed into a 1.5 mL microfuge tube containing either (i) 1 mL of blue tap water or 1 mL of blue absolute ethanol, or (ii) 1 mL of red dimeticone/cyclomethicone. Lice were kept immersed for 10 min, 30 min or 12 h. Controls consisted of 9 groups of lice (n = 3 each), which were processed identically, except for the absence of dye. After immersion, lice were dried gently on a filter paper to absorb excess of fluid and then abundantly rinsed with tap water before examination under a binocular magnifier.p < 0.05 level using JMP version 10.0.0 .The survival of lice after immersion or anoxia bioassays was expressed as the percentage of living specimens. For immersion and anoxia bioassays, the Fisher’s exact test was used to compare the survival rate between groups, looking for a relationship with time of immersion or exposure by the Cochran-Mantel-Haenszel tests. For anoxia bioassay, the 3-way ANOVA model with interaction was used to check whether the treatment (anoxia or control) remains independent of the time of exposure. The analyses were carried out at the two-tailed p < 0.0001). After H10, death was observed in the anoxic group  of lice were alive compared with 87.1% (305/350) in the matched control groups  of water-immersed lice were alive. Mortality was higher in the control group compared with the test group . The dynA total of 36 lice were aliquoted into 6 study groups (n = 3 each) and 6 untreated control groups (n = 3 each). Irrespective of the specific experiment (water-immersed or non-immersed) and the freezing method (−20 °C or −196 °C), the diameter of the thoracic and abdominal spiracles measured approximately 20 μm a and ∼5 A total of 54 head lice were used for this study. All lice immersed in water containing stain Figure 4i, j, k) showed a total lack of coloration of the tracheal system regardless of the duration of contact . On the other hand, colored ethanol stained the lice spiracles within 10 min of contact . The fact that after 14 h of anoxia, only 12.2% of lice were alive compared with 48.9% of controls demonstrates that either long-term oxygen deprivation or carbon dioxide increases, ranging from 8 to 16% (after 24 h /or both) kill lice. The AnaeroGenTM sachet reduces oxygen to < 0.1% within 2.5 h, while generating 8–16% carbon dioxide, suggesting that lice are adapted to total oxygen deprivation for 10-12 h. Death of lice by asphyxiation was claimed for several physically active treatments [TM pouch where carbon dioxide levels never exceeded 16%. After 16 h of anoxia, the death rate was very high. A limitation of our study was that controls were maintained in an ambient atmosphere rather than in anaerobic pouches, i.e. without use of the AnaeroGenTM sachet. Thus, the high mortality rate in the untreated controls during the first few hours could be due to dehydration.Data to quantify how long head lice can survive under anoxic conditions are lacking. This study showed that oxygen deprivation caused higher mortality rates of all groups subjected to this condition for >8 h, when compared with untreated controls  [et al. [et al. [When immersed in water, we observed a survival rate of 100% after 6 h, and 50% after 24 h; during these experiments louse mobility ceased totally. The latter observation had also been reported in previous studies with head lice  and amphcrochir) . Our res [et al.   that results in instant freezing of the physical state of spiracles during immersion. Interestingly, modification of the diameter of spiracles through slow or instant closing was not observed in our study. There was no difference in the size of spiracle diameter when lice were in the air or in water; prevention of water penetration probably relies on an alternative mechanism. This contradicts previous reports, which on examination brought little insight on the mechanism itself ,12,31,33As far as we are aware, water ingress through spiracles during the immersion process has never been demonstrated. In our study, although all water-immersed head lice showed open spiracles, colored water did not penetrate into the respiratory system i-k. Howeet al. with benzyl alcohol [In contrast with what was observed following water immersion, the respiratory system of lice immersed in ethanol was flooded within half an hour b. This i alcohol ; however alcohol ,47,50. I alcohol .®) as Burgess [Dimeticone/cyclomethicone appears to have entered through the spiracles to flood the respiratory system in a similar manner, which results in head lice death within 30 min. Death can be explained by (i) the impossibility for head lice to expel dimeticone/cyclomethicone out of the respiratory system, whereby the insect dies by suffocation as previously reported , or (ii) Burgess , and our Burgess  (Figure In this study, experiments were performed on head lice collected from several patients from various ethnic communities and the therapeutic history of each patient was not known.To conclude, a better understanding of the environmental adaptation of lice to the human host may allow us to find improved methods to control head lice.In this study, we showed that head lice were capable of physiological or behavioral adaptations for surviving anoxia and immersion in water. They can endure anoxia for several hours and are resistant to immersion in water without obvious deleterious effects on movements and survival. Such findings must be taken into account to provide innovative and effective countermeasures against head lice infestation. It appears that efficacy is related to physical entry into the respiratory system that can be easily monitored using dyes.The authors declare that they have no competing interests.