Nebulized antibiotic therapy directly targets airways and lung parenchyma resulting in high local concentrations and potentially lower systemic toxicities. Experimental and clinical studies have provided evidence for elevated lung concentrations and rapid bacterial killing following the administration of nebulized antibiotics during mechanical ventilation. Delivery of high concentrations of antibiotics to infected lung regions is the key to achieving efficient nebulized antibiotic therapy. However, current non-standardized clinical practice, the difficulties with implementing optimal nebulization techniques and the lack of robust clinical data have limited its widespread adoption. The present review summarizes the techniques and clinical constraints for optimal delivery of nebulized antibiotics to lung parenchyma during invasive mechanical ventilation. Pulmonary pharmacokinetics and pharmacodynamics of nebulized antibiotic therapy to treat ventilator-associated pneumonia are discussed and put into perspective. Experimental and clinical pharmacokinetics and pharmacodynamics support the use of nebulized antibiotics. However, its clinical benefits compared to intravenous therapy remain to be proved. Future investigations should focus on continuous improvement of nebulization practices and techniques. Before expanding its clinical use, careful design of large phase III randomized trials implementing adequate therapeutic strategies in targeted populations is required to demonstrate the clinical effectiveness of nebulized antibiotics in terms of patient outcomes and reduction in the emergence of antibiotic resistance. Effective antimicrobial therapy requires adequate drug concentrations at the site of the infection. This is often not possible when using intravenous therapy among intensive care unit (ICU) patients who require mechanical ventilation due to altered pharmacokinetics and poor lung tissue penetration of many antimicrobial agents , 2. OutcThe present review summarizes current practical constraints for optimal delivery of nebulized antibiotics to the lung parenchyma during invasive mechanical ventilation, and the resulting pharmacokinetics and pharmacodynamics. Current clinical practice is put into perspective with evidence that has become available from recent clinical studies so as to provide a better understanding of the relevance of future phase III trials.Delivery of high concentrations of antibiotics to infected lung regions is the key to achieving efficient nebulized antibiotic therapy. The antibiotic dose placed in the nebulizer should take into account the significant extrapulmonary drug deposition . Poor implementation may result in extrapulmonary deposition as high as 97% [The optimal mass median aerodynamic diameter that allows for distal lung deposition ranges from 0.5 to 3 µm . ParticlTable Medication dilution and the nebulizer fill volume influence particle size and drug delivery. For a given dose, a larger fill volume with a diluted solution can overcome the residual volume issues mentioned above . NeverthY-piece [Y-piece depends on bias flow and the circuit section. Indeed, the bias flow flushes aerosol into the expiratory limb during expiration, inducing aerosol loss  following intravenous infusion of 7–10 mg/kg of this drug . Even whFrom a pharmacodynamic point of view, aminoglycosides and colistin are concentration-dependent antibiotics with a post-antibiotic effect. They are particularly suitable for nebulization as high lung concentrations can be expected and only 1 to 3 daily administrations are required. Time-dependent antibiotics, such as β-lactams or glycopeptides, require drug concentrations to be maintained above the MIC throughout the dosing interval. Continuous or closely repeated administration is hence required , 34, whiNephrotoxicity associated with CMS and aminoglycosides after intravenous infusion represents an additional rationale for their nebulized delivery. Given the lack of a proven benefit and increased nephrotoxicity, a recent Cochrane systematic review discouraged the use of intravenous aminoglycoside in combination with β-lactam antibiotics for treating sepsis . ConversThese theoretical favorable pharmacokinetic and pharmacodynamic profiles, summarized in Fig. Escherichia coli. The amikacin concentrations measured in the infected lung parenchyma were significantly higher than the MIC and 3–30 times higher than after intravenous infusion [Pseudomonas aeruginosa. Peak concentrations in the infected lung parenchyma were significantly higher than the MIC [The efficacy of 45 mg/kg/24 h nebulized amikacin has been studied in ventilated piglets with pneumonia due to infusion . The eff the MIC . Improve the MIC .These experimental studies, documenting high lung parenchymal antibiotic concentrations after nebulization of high doses and implementing optimized techniques, laid the foundations for clinical pharmacokinetic studies.It was recently shown that in patients with healthy lungs, 10–15% of the nebulizer charge is deposited in the lungs . Such a In patients with VAP, nebulized amikacin 400 mg/12 h) achieved median lung ELF concentrations 100 times higher than the maximum serum concentration 0 mg/12 h measuredIn patients with VAP or ventilator-associated tracheobronchitis (VAT) who were administered 1 million international units  of CMS via a vibrating mesh nebulizer under optimal conditions every 8 h, peak ELF concentrations were high but then dropped below the sensitivity breakpoint at 4 h, thus indicating that this dose may not be optimal for treating pneumonia  Table 22. NebuliP. aeruginosa as compared to intravenous therapy [One randomized controlled trial studied the efficacy of nebulized antibiotic therapy only to treat VAP caused by  therapy . Forty pTesting another clinical strategy, Palmer et al.  assessedThe systemic antibiotic sparing effect of nebulized antibiotics was also observed in the earlier mentioned work by Niederman et al. . For 69 In these studies, various doses of nebulized amikacin were used (twice as many high-dose versus low-dose studies) and aerosols were delivered with different nebulizers although close attention was often paid to optimize the nebulization technique. Nebulized amikacin allowed for effective treatment of bronchial and parenchymal infections, even when involving bacteria with high MIC, and it consistently reduced the emergence of resistant bacteria. Clinical cure, evaluated as a secondary endpoint in these trials, depends in part on the therapeutic efficacy in the control group. Further clinical phase III studies are required to prove the clinical benefit of nebulized amikacin.P. aeruginosa and Acinetobacter baumannii [As colistin is the most frequently nebulized antibiotic in ICUs, there is some information in regard to clinical bedside safety and efficacy as well as large retrospective databases. However, only a small number of prospective controlled studies have evaluated nebulized colistin in mechanically ventilated patients implementing optimized technique. High-dose nebulized CMS (5 MIU/8 h with strict optimized technique) has been evaluated in patients with VAP caused by multidrug-resistant (MDR) aumannii . Patientaumannii . A recenaumannii , althougaumannii .Three groups of investigators used databases with information regarding patients suffering from colistin-only susceptible bacterial VAP to evaluate whether nebulized CMS as adjunct to intravenous CMS is beneficial. One study observed no additional benefit of combined nebulized and intravenous CMS therapy , whereasMeta-analyses of the clinical studies have yielded conflicting results and further clinical evidence from randomized trials is required, while more extensive evaluation of renal toxicity related to the administration of high dose of CMS or amikacin intravenously or by nebulization is also needed –65. The Aside from investigating curative nebulized antibiotics to treat patients suffering from VAT and/or VAP, some authors have also tested nebulized colistin, ceftazidime or aminoglycosides for prophylaxis in intubated patients. Two small-sized studies obtained positive results with such a preemptive nebulized therapy in terms of the VAP frequency, and they also observed no significant change in the bacterial antibiotic sensitivity pattern , 67. FurThe spread of MDR associated with the favorable data outlined above led to implementation of nebulized antibiotic therapy in the clinical setting despite the lack of large-scale patient-centered evidence. Among 816 international intensivists surveyed electronically, one-third reported that they usually or frequently nebulize colistin . In an oThis practice pattern challenges the conclusions drawn from some prospective and retrospective studies without standardized nebulization procedures. In many patients, the amount of drug delivered beyond the tip of the endotracheal tube may be negligible, and low drug pulmonary concentrations may enhance the selection of drug-resistant bacteria. The observed practices illustrate the difficulties of implementing optimal nebulization techniques in patients outside of controlled clinical research settings, and this may have implications for future trial designs and the dissemination of knowledge.In order to guarantee adequate safety and aerosol therapy efficiency during mechanical ventilation, standard operating procedures should be implemented including a checklist for physicians and nurses , 72. AdeA filter needs to be positioned between the expiratory limb and the ventilator to protect the latter from expired particles and to prevent dysfunction Fig. . A new fLong-term bronchial toxicity and alveolar damage that can result from high local antibiotic concentrations have received scant attention. Whereas a transient benign cough is common, bronchospasm is a more severe, albeit infrequent, side effect that has been reported to occur during antibiotic nebulization , 34, 40.Circuit manipulation for nebulization must follow the usual hygiene standards; the availability of single-patient-use nebulizers contributes to hygiene control. Desaturation and hypoxemia have been reported in patients receiving frequent repeated nebulization  due to aBronchospasm and obstruction of expiratory filters are first detected as an increase in the peak airway pressure. These complications emphasize the need for close monitoring of the peak airway pressure and oxygenation during nebulization . SystemiSince 2007, six meta-analyses have been published in regard to nebulized antibiotics as a treatment for lung infections among ventilated patients –65, and Patients with a high rate of intravenous treatment failure are most susceptible to benefit from this approach. Thus, patients and/or ICUs at high risk of the emergence of MDR bacteria represent target populations for nebulized antibiotics. Defining populations at high risk of toxicity (mainly patients with acute kidney injury) may also be a worthwhile challenge.Intravenous therapy is effective and well tolerated in most patients with VAP caused by β-lactam-susceptible bacteria. The benefit of withholding this therapy is elusive. In patients with late-onset VAP caused by difficult-to-treat bacteria with frequent recurrence, in the light of the questionable benefit of intravenous aminoglycosides, nebulized aminoglycosides as adjunct to systemic therapy may be considered , 81. ForThis is urgently needed to implement easy, safe and reproducible techniques as well as to standardize nebulization practices for clinical trials and to thereafter translate the results into clinical practice. A paradigm change may occur in the future with the development of inhaled anti-infective nanoparticle antibody or phage therapies.Experimental and clinical pharmacokinetics and pharmacodynamics support the feasibility and possible benefits of nebulized antibiotic therapy to treat VAP in mechanically ventilated patients. Before expanding its clinical use, optimization of nebulization techniques and standardization of nebulization procedures are urgently needed. Large phase III randomized trials are required to demonstrate the clinical effectiveness and benefits in terms of improvements in patient outcomes and reduction in the emergence of antibiotic resistance.


Different leaching trends were observed via X-ray fluorescence when comparing chemical with biological leaching. Lower leaching rates were found for concrete samples fortified with silica fume than those without silica fume. X-ray diffraction and scanning electron microscopy confirmed a massive sulfate precipitate formation on the concrete surface due to bacterial exposure.Current design practices based on descriptive approaches to concrete specification may not be appropriate for the management of aggressive environments. In this study, the durability of cement-based materials with and without the addition of silica fume, subjected to conditions that leach calcium and silicon, were investigated. Chemical corrosion was simulated by employing various H As repoica fume . Similarica fume . Howevert pastes .4+ than Ca2+ leaching were detected for all samples subjected to bacterial attack. A comparison of the Si4+ and Ca2+ leaching rates due to H2SO4 (pH 4) attack and a bacterial medium with the same pH of 4 revealed that the bacterial attack was more aggressive.Higher rates of SiTo confirm the superior performance of silica fume-based concrete in an aggressive sulfate environment, the mass percentage of the dissolved ions  was also4+ and Ca2+ leachability was confirmed for all concrete samples with the exception of samples immersed in fresh water. The most significant difference was noticed for samples subjected to bacterial attack. The calculated leachable fraction of Si4+ was 5.2- and 3.3-fold higher for samples without silica fume compared with the samples with silica fume after bacterial inoculation. Significantly lower leachable fractions of both Si4+ and Ca2+ ions were also observed for silica fume-based samples exposed to H2SO4 with a pH of 3 (2.9-fold for Si4+ and 2.3-fold for Ca2+).The superior performance of concrete samples based on silica fume in terms of both SiThe results of the mass changes of the analyzed concrete samples prior to and after the experiments are given in A decrease in mass was noticed for all concrete specimens after the chemical corrosion experiments, whereas an increase in mass was detected for all samples after the biological corrosion experiments, as can be observed in A histogram of mass changes of the concrete specimens with and without the addition of silica fume prior to leaching and after 270 days of leaching is shown in 2SO4 with a pH of 3, which corresponds with the findings regarding the leaching of Si4+ and Ca2+. Surprisingly, higher mass changes were found for all concrete samples with the addition of silica fume than samples without silica fume. As is known, the deterioration of concrete can be caused by both mechanisms: (i) a dissolution of the cement paste constituents and its subsequent removal from the paste matrix due to its inherently high solubility and (ii) chemical reactions within the paste, e.g., salt crystallization, resulting in concrete volume expansion. The decrease in mass is linked with the leaching process, whereas an increase in mass can be linked with the penetration of sulfate solutions either by simple diffusion or capillary suction, which causes some salts to undergo cycles of dissolution and crystallization. The mass changes after chemical exposure indicate that the leaching process dominates in silica fume-based samples, whereas the crystallization process dominates in concrete samples without silica fume. The percentage of mass changes for concrete samples varied from 0.12% (sample M0-3) to 2.42% (sample M1-1). The highest decrease in concrete mass was detected for samples exposed to the most aggressive environment, represented by HThe formation of easily visible corrosion-induced cracks on the surface of concrete samples exposed to different aggressive media has been observed . No sign2SO4 with a pH of 4 (M0-2 and M1-2 samples) and concentrated bacterial medium with a pH of 4 (M0-6 and M1-6 samples) were also analyzed using SEM and EDX, similar to our previous work [42− (gypsum and thaumasite) was observed via SEM 6(CO3)(SO4)·12H2O) and gypsum (CaSO4·2H2O) can be assumed to be acting on the concrete surfaces. As reported by Schmidt et al. [42−.As can be observed in t et al. , despite2O3, 18.11 SiO2, 0.33 P2O5, 1.49 SO3, 0.06 Cl, 1.12 K2O, 57.15 CaO, 0.18 TiO2, 0.001 MnO, and 2.70 Fe2O3. Basic components of silica fume , representing a mineral waste admixture, measured using XRF in wt % were as follows: 1.07 MgO, 0.36 Al2O3, 92.46 SiO2, 0.05 P2O5, 0.07 SO3, 0.14 Cl, 0.99 K2O, 0.23 CaO, 0.58 MnO, and 1.24 Fe2O3.The concrete samples used in the experiment were prepared using cement CEM I 42.5 N . The basic chemical composition of cement measured using XRF  in wt % was as follows: 1.37 MgO, 4.02 Al3, consisting of cement (Povazska cementaren), silica fume (OFZ a.s.), water, aggregate  with particle size fractions of 0/4 mm, 4/8 mm, and 8/16 mm, and plasticizer Stachement 2353 based on polycarboxylates  were employed (Two mixtures (M0 and M1), per memployed . Accordi−3 (M1). Slightly different water to cement ratios of M0 and M1 concrete mixtures were designed to maintain consistency (Slump S2) in the mixtures. Standardized concrete prisms measuring 100 × 100 × 400 mm3 were poured and cured for 28 days immersed in water at the ambient temperature of 20 °C prior to testing [3 for corrosion testing. They were slightly brushed to remove loose particles, sterilized in 70% ethanol for 24 h to disinfect the concrete surfaces before the bacterial experiment, and dried at 105 °C until a constant mass was reached. Constant mass was achieved when less than 0.1% of the test sample wet mass is lost during additional exposure to the drying process.Mixtures M0 and M1 were designed to meet the requirement of at least C30/37 concrete strength class and a water-cement ratio (w/c) of a maximum of 0.5 in accordance with EN 206-1. The concrete samples met the criteria for exposure class XA2  with wat testing ,40. The 2SO4, pH 3.0 , and 0.0005 wt % H2SO4, pH 4.0 , in glass beakers, both being daily checked for pH  and kept constant by the addition of 0.1 M H2SO4. Other prisms were immersed into 400 mL of MgSO4 solution of 12.5 g·L−1 with SO42− 10 g·L−1 (samples M0-3 and M1-3), an MgSO4 solution of 3.75 g·L−1 with SO42− 3 g·L−1 (samples M0-4 and M1-4), and fresh water with a pH of 7.2 (samples M0-5 and M1-5) for concrete corrosion resistance testing [2+ and Si4+ were measured in leaching media.The latter concrete prisms were exposed by immersion into 400 mL of 0.005 wt % H testing . The volA. thiooxidans was isolated from an acid mine drainage  as described by Waksman and Joffe [A culture of nd Joffe .4+ and Ca2+ were measured in the leachates every 30 days.The concrete prisms were exposed at 23 °C in covered glass jars to 400 mL of a bacterial culture medium consisting of 20 vol. % of bacterial inoculum and 80 vol % nutrient medium with pH 4.0 according to Waksman and Joffe (samples M0-6 and M1-6) or the same medium diluted with distilled water 1:1 (samples M0-7 and M1-7). Six milliliters of bacterial culture was inoculated at 7-day intervals over a period of 270 days. The concentrations of dissolved SiThe concentrations of Ca and Si in the leachates were determined using XRF  and a silicon drift detector with a resolution of 145 eV at 10,000 pulses. The primary beam was polarized with a Bragg crystal and highly ordered pyrolytic graphite target. Measurements were performed for 300 and 180 s at voltages of 25 kV and 50 kV and currents of 0.5 and 1.0 mA in the presence of helium.−2; this was analyzed using XRF.To determine the chemical composition prior to and after the experiments, cement composites were crushed into powder , and 5 g was mixed with 1 g of a dilution material  and subsequently pressed into a pellet at a pressure of 0.1 MPa·mα radiation generated at 10 mA and 30 kV with a step size of 0.04° over the range 2θ from 10° to 90°.The precipitates on the surface of the concrete samples after the experiments were removed and placed on adhesive carbon tape and studied using SEM/EDX  and XRD  with Cu KAcidithiobacillus thiooxidans was more dangerous, in terms of leaching of the main components, than chemical sulfate attack with the exception of a H2SO4 solution of a pH of 3.Microbiological attack precipitated by 4+ and Ca2+, than concrete samples without silica fume.The concrete samples equipped with silica fume exhibited better durability, in terms of the leaching of Si4+ and Ca2+, respectively, were observed for silica fume-based samples exposed to H2SO4 with a pH of 3 compared with samples without silica fume.Significantly lower leachable fractions, specifically those 2.9- and 2.3-fold lower for Si4+ was 5.2- and 3.3-fold higher for samples without silica fume compared with the samples with silica fume under biological exposure.The calculated leachable fraction of SiSilica fume concrete is not impervious to all aggressive chemicals. However, the study shows that at a low water to cement ratio (w/c), silica fume concrete can effectively prevent significant damage following many types of chemical attack including sewage leachate. Based on this finding, silica fume concrete has been specified for use in sewer and outfall pipes.This paper presents the results of a study on the deterioration of concrete samples caused by chemical and microbiological attacks. Attention was paid to the leachability of the most important components of the cement matrix: Si(IV) and Ca(II) compounds. The mass and surface changes due to this deterioration were of interest as well. The following conclusions have been obtained:Notably, many problems related to both the chemical and microbial corrosion of concrete have not been solved up to now despite considerable progress in the recognition of corrosion kinetics and mechanisms. Thus, further research on corrosion processes and methods of protection against these processes is necessary.


The General Medical Council (GMC) conducts Tests of Competence (ToC) for doctors referred for Fitness to Practise (FtP) issues. GPs take a single best answer knowledge test, an Objective Structured Clinical Examination (OSCE), and a Simulated Surgery (SimSurg) assessment which is a simulated GP consultation. The aim of this study was to examine the similarities between OSCEs and SimSurg to determine whether each assessment contributed something unique to GP ToCs.A mixed methods approach was used. Data were collated on 153 GPs who were required to undertake a ToC as a part of being investigated for FtP issues between February 2010 and October 2016. Using correlation analysis, we examined to what degree performance on the knowledge test, OSCE, and SimSurg related to case examiner recommendations and FtP outcomes, including the unique predictive power of these three assessments. The outcome measures were case examiner recommendations (i) not fit to practise; ii) fit to practise on a limited basis; or iii) fit to practise) as well as FtP outcomes (i) erased/removed from the register; ii) having restrictions/conditions; or iii) be in good standing).For the qualitative component, 45 GP assessors were asked to rate whether they assess the same competencies and which assessment provides better feedback about candidates.p < 0.001. SimSurg had additional predictive power in the presence of OSCEs and the knowledge test (p = 0.030) in distinguishing doctors from different FtP categories, while OSCEs did not (p = 0.080). Both the OSCEs (p = 0.004) and SimSurg (p < 0.001) had significant negative correlations with case examiner recommendations when accounting for the effects of the other two assessments.There was significant overlap between OSCEs and SimSurg, Inductive thematic analysis of the responses to the questionnaire showed that assessors perceived OSCEs to be better suited to target specific knowledge and skills. SimSurg was thought to produce a more global picture as the scenarios more accurately portray a patient consultation.While all three assessments are strong predictors of both case examiner recommendations and FtP outcomes, our findings suggest that the efficiency of GP ToCs can be improved by removing some of this overlapping content. The General Medical Council (GMC) holds the right to assess the fitness to practise (FtP) of doctors on the medical register in the UK . In 2015Part of the GMC investigation into doctors who are referred for fitness to practise following concerns around their performance at work may include a Test of Competence (ToC) . The ToCThe results of the ToC assessments are used alongside the results obtained from a peer review exercise to help the assessment team write a report that includes a recommendation about the doctor. This recommendation is then reviewed by two case examiners  who can conclude the case with no further action, issue a warning, agree undertakings with the doctor, or refer the case to the Medical Practitioners Tribunal Service (MPTS) for a hearing .without warnings or conditions, the doctor remaining on the register with warnings/conditions, or the doctor being erased from the register [MPTS is a statutory committee of the GMC that has independent decision-making authority, and is responsible for adjudicating FtP cases . The triregister .General practice is the specialty that receives most complaints: they were involved in 43% of all complaints in 2015 . The seqThe overall aim of this study was to examine the similarities between OSCEs and SimSurg to quantitatively determine whether each assessment contributed something unique to GP ToCs, and to assess the perceived benefits and limitations of both modalities from a qualitative perspective.Examine the relationship between each assessment and FtP outcomes/case examiner recommendations.Examine the correlations between FtP outcomes and case examiner recommendations.Examine the unique predictive power of each assessment in terms of FtP outcomes and case examiner recommendations.Explore assessors’ opinions of OSCEs and SimSurg and how well these correspond to the quantitative findings.Specific objectives:Mixed method research methodology involves the integration of both qualitative and quantitative research designs . We tookWe examined quantitative data from doctors undergoing GP ToCs conducted between February 2010 and October 2016, identified through the GMC register of doctors that have been investigated for fitness to practise purposes. For the qualitative component, the GMC training team contacted all of the 45 assessors that had performed a GP assessment in the past 18 months. They were sent an email with a link to the questionnaire. The email explained ‘the aim of the study is to gather your views about these assessment methods in order to ensure our GP ToC assessments are efficient and remain of high standards’.i)not fit to practise;ii)fit to practise on a limited basis;iii)fit to practise.We examined the ability of each assessment to predict case examiner recommendations and FtP outcomes. The possible case examiner recommendations were:i)erased/removed from the register ;ii)having restrictions/conditions on the GMC registration;iii)or be in good standing without conditions.The possible FTP outcomes were:Performance assessmentFor the qualitative component, we asked GP assessors who were involved in conducting ToCs between September 2014 and March 2016 about the utility of OSCEs and SimSurg in assessing GP’s fitness to practice.The 120 item SBA knowledge test chosen specifically for each doctor under investigation was computer marked, with a mark out of 120 converted to a percentage for reporting. The 12 bespoke OSCE stations chosen for each doctor were marked out of 40, as well as using the categories ‘acceptable’ ‘cause for concern’ and ‘unacceptable’. The 10 SimSurg assessments prior to October 2014 were in the form of a set roster of stations of which there were two rosters (first sit and resit assessment), after October 2014, this was changed to 10 bespoke stations. Due to these differences, and the bespoke nature of most assessments, only total scores in each assessment as a percentage of the total score has been used in the data analysis.2)Assessors’ opinionsWe performed between-subjects ANOVA to look for group differences. We used Pearson’s r correlation to examine the overlap between the assessments with each assessment as a predictor where FtP outcome and case examiner recommendation were dependent variables. We used Spearman’s correlation to examine the overlap in the FtP outcome categories and case examiner recommendation. Fisher’s transformations were conducted to examine the unique contribution of each assessment to FtP outcomes and case examiner recommendations. All analyses were conducted using SPSS version 22.“Are there benefits of using OSCEs over SimSurg?” and “Are there benefits of using SimSurg over OSCEs”. We also asked respondents to rate agreement with statements about whether the two modalities assess the same competencies, and which provides better feedback about candidates’ performance. We performed inductive thematic analysis on the free text responses based on the method described by Braun and Clark [AS, KK, CV, LM developed a short 10–15 min survey using UCL’s secure online questionnaire platform, Opinio. The survey ran from 23 March to 19 May 2016. All responses were anonymous. The questions were formulated through team discussion and the final version was reviewed and approved by AS, KK, CV, and LM. Respondents were asked to express views regarding OSCE and SimSurg in free text box responses. The questions posed for this free text responses were: nd Clark . SpecifiOverall, 153 GPs took a ToC between February 2010 and October 2016. For the qualitative component, 34 of the 45 GP assessors responded, giving a response rate of 76%.The demographics of the GPs are summarised in Table In terms of the final FtP outcomes, 63 of the 153 GPs were no longer on the GMC register , 50 were on the register with warnings, conditions, suspensions, and/or undertakings, while 40 were on the register in good standing.Average scores on the SBA knowledge test, OSCEs and SimSurg for GPs in each of the three outcome groups are shown in Table p < 0.001), and GPs on the register in good standing (p < 0.001). There were no significant differences in any of the assessment scores when comparing GPs on the register with warnings/sanctions compared to GPs on the register in good standing (p > 0.05).Specifically, for the knowledge test score, OSCEs and SimSurg, post-hoc contrasts showed that GPs removed from the GMC register scored significantly lower than GPs with warnings/sanctions (p > 0.05). There were also significant positive correlations between the assessments with moderate to large effect sizes p < 0.001. As can be seen in Table There were significant negative correlations between the outcomes of FtP ToCs and all the assessments . However, the relationship between OSCEs and FtP outcomes was no longer significant when accounting for the effect of SimSurg and the knowledge test . That is, while all three assessments were significant predictors of FtP outcomes, there was significant overlap in content, such that OSCEs did not provide additional predictive power when taking into account the effect of SimSurg and the knowledge test. In contrast, SimSurg did provide additional predictive power.Due to the significant correlation between the three assessment measures, partial correlations were conducted to examine the contribution of each assessment to FtP outcomes, while controlling for the effect of the other two assessments. There was a significant negative correlation between FtP outcomes and SimSurg, when accounting for the effect of OSCEs and the knowledge test  and also compared to GPs recommended as fit to practise generally (p < 0.001). There were no significant differences in the knowledge test scores when comparing GPs recommended as fit to practise on a limited basis compared to those recommended as fit to practise generally (p > 0.05). However, GPs who were classified as fit to practise on a limited basis performed significantly lower on the OSCEs and SimSurg compared to those classified as fit to practise generally .For all three assessments, post-hoc contrasts showed that GPs recommended as not fit to practise performed significantly poorer than GPs recommended as fit to practise on a limited basis .There were significant negative correlations between the case examiner recommendations and all the assessments, with moderate to large effect sizes, ρ = −0.33, p < 0.001). There was also a smaller, significant negative correlation between case examiner recommendations and OSCEs, when accounting for the effect of SimSurg and the knowledge test .Due to the significant overlap between the OSCEs and SimSurg, partial correlations examining the contribution of each assessment to case examiner recommendations, while controlling for the effects of the other assessments were conducted. There was a significant negative correlation between case examiner recommendations and SimSurg, when accounting for the effect of OSCEs and the knowledge test  scored poorly compared to those that remained on the register with warnings/sanctions, as well as compared to those that remained on the register in good standing. For case examiner recommendations, GPs recommended to be erased from the register performed poorly compared to those recommended as fit to practise on a limited basis, as well as compared to those recommended as fit to practise. Further, there was significant overlap in the doctors categorised by FtP outcomes and case examiner recommendations, highlighting the consistency between the two FtP decision making stages. In terms of the demographics of doctors under investigation, these doctors tended to be male, trained outside of the UK, and obtained their medical qualification prior to 1992, consistent with doctors under investigation in prior research , 13.When looking at the relationship between assessments and outcome categories, all three parts of the assessment were significantly related to outcomes of FtP investigations and case examiner recommendations, and on their own, there were no substantial differences in the predictive ability of the three components. However, there was overlap between the assessments, particularly between OSCEs and SimSurg.Overall, assessors perceive both OSCE and SimSurg modalities to be valid and with their own merits. They thought that OSCEs allow for specific skills testing in non-consultation settings, whereas SimSurg provides an overall impression of GP ability and has higher face validity for clinical practice. Nonetheless, the assessors thought that both modalities deserved inclusion in future ToCs. However, as SimSurg assesses many of the same competencies as the OSCE, assessors thought a reduction from 12 to six OSCE scenarios is a potential modification to future GP ToC assessments.Our findings indicate that as stand-alone assessments, all three assessment modalities are good discriminators of various FtP investigation outcomes and case examiner recommendations, with performance in all three assessments related to final FtP ToC outcomes and case examiner recommendations in a similar manner. This is consistent with previous research demonstrating the reliability and validity of the FtP ToCs –5, 14.and case examiner recommendations. OSCEs on the other hand, did not have additional predictive power of FtP outcomes in the presence of SimSurg and the knowledge test. This is consistent with the view of GP assessors, who reported SimSurg as being the assessment modality that has the higher face validity and that provides a better overall impression of a GP’s ability.Moreover, we found that SimSurg had additional predictive power in the presence of OSCEs and the knowledge test score for both FtP outcomes These findings suggest that there may be an opportunity to increase the efficiency of GP ToCs by removing either the OSCE or SimSurg components. We recommend the removal of OSCEs and retaining of SimSurg in future assessments. This is because, while they were both strong predictors of FtP outcomes and case examiner recommendations, SimSurg had additional predictive value, and was reported to have higher face validity by GP assessors. This modification is further warranted as it would reduce the length of the assessment for the candidates and reduce assessor workload, while being unlikely to compromise on the validity of the GP ToCs .Our findings also have wider implications for other medical assessment settings where OSCEs are used for workplace assessment, as used in many board examinations not only in the UK but also in other Western countries such as Canada and Australia. Specifically, our findings suggest that the limitations of OSCEs  may be reduced/overcome by the use of SimSurg, which requires an integration of multiple competencies similar to real-world medical practise. Indeed, a Canadian study examined the validity of using a hybrid simulation-OSCE assessment setting in a group of urology residents and found that the incorporation of SimSurg is likely to be a more valid assessment of trainee skills, compared to the use of OSCEs alone . FurtherThere are several limitations that need to be considered when interpreting the results of this study. First, due to the bespoke nature of the ToC assessments , 5, it iIn terms of the limitations of the qualitative component, 30% of GP assessors that were asked to participate in the survey declined participation, and we were not able to ascertain reasons for this. Hence, there may be some response bias in the opinions expressed by the GP assessors who participated. Further, GP assessors were of the opinion that a reduction in OSCE stations from 12 to six would yield a valid assessment at the same time as retaining OSCE stations that tested different skills to those in SimSurg. However, it was beyond the scope of this study to determine the reliability and validity of such a modification.unique predictor of both FtP outcomes and case examiner recommendations compared to the OSCEs, although this difference was not statistically significant. This is consistent with the view of GP assessors who reported that SimSurg provides an overall impression of GP ability and has higher fidelity to clinical practice. Taken together, these findings are important in the context of increasing the efficiency and predictability of future GP ToCs and should be considered as preliminary evidence, supporting a modification in ToCs to remove the OSCE component and retain the SimSurg component. Further investigation, with consideration to factors such as time since primary medical qualification, country of primary medical qualification, and gender, are needed to further examine the effect of demographic factors on potential changes to GP ToCs in the future.Notwithstanding these limitations, this study is the first to examine the utility of each GP ToC assessment modality in predicting FtP investigation outcomes concurrently with case examiner recommendations. It also provides a qualitative investigation of GP assessor’s opinion on the utility of OSCEs and SimSurg assessments. Findings from this study indicate that when examined alone, all three assessments are related with final FtP ToC outcomes and case examiner recommendations. That is, all three assessments are strong predictors of both case examiner recommendations and FtP outcomes, demonstrating their validity in the ToC assessment setting. However, our work also demonstrates that there is a lot of overlap between the assessments, particularly between the OSCEs and SimSurg. When this overlap in content is taken into account, SimSurg was a marginally better


The concept of ventilator-associated tracheobronchitis is controversial; itsdefinition is not unanimously accepted and often overlaps withventilator-associated pneumonia. Ventilator-associated tracheobronchitis has anincidence similar to that of ventilator-associated pneumonia, with a highprevalence of isolated multiresistant agents, resulting in an increase in thetime of mechanical ventilation and hospitalization but without an impact onmortality. The performance of quantitative cultures may allow better diagnosticdefinition of tracheobronchitis associated with mechanical ventilation, possiblyavoiding the overdiagnosis of this condition. One of the major difficulties indifferentiating between ventilator-associated tracheobronchitis andventilator-associated pneumonia is the exclusion of a pulmonary infiltrate bychest radiography; thoracic computed tomography, thoracic ultrasonography, orinvasive specimen collection may also be required. The institution of systemicantibiotic therapy does not improve the clinical impact of ventilator-associatedtracheobronchitis, particularly in reducing time of mechanical ventilation,hospitalization or mortality, despite the possible reduced progression toventilator-associated pneumonia. However, there are doubts regarding themethodology used. Thus, considering the high prevalence of tracheobronchitisassociated with mechanical ventilation, routine treatment of this conditionwould result in high antibiotic usage without clear benefits. However, wesuggest the institution of antibiotic therapy in patients with tracheobronchitisassociated with mechanical ventilation and septic shock and/or worsening ofoxygenation, and other auxiliary diagnostic tests should be simultaneouslyperformed to exclude ventilator-associated pneumonia. This review provides abetter understanding of the differentiation between tracheobronchitis associatedwith mechanical ventilation and pneumonia associated with mechanicalventilation, which can significantly decrease the use of antibiotics incritically ventilated patients. VAT is often considered an alternativediagnosis of ventilator-associated pneumonia (VAP).The definition of ventilator-associated tracheobronchitis (VAT) is not consensual. Inpatients ventilated for more than 48 hours, fever with no other cause and/orleukocyte count > 12,000/µL or < 4.000/µL associated withincreased volume and/or purulence of respiratory secretions and endotrachealaspirate (ETA) with bacterial growth in the absence of a new pulmonary infiltrate orprogression of previous infiltrate define this condition.In this article, we review the published work on the use of antibiotic therapy inVAT. A search on the PubMed database was performed with the terms "ventilator","tracheobronchitis", and "antibiotic" without time limitations. Manual search ofrelevant references cited in the selected papers was also performed. Only articlesin English were considered.,4,6-8 In a recent meta-analysis with 3,362 patients,the incidence of VAT was 11.5%, ranging between 1.4% and16.7%. According to the literature, it is possible toprogress from VAT to VAP,,8,10,11 and this phenomenon is reported in12.2% to 34% of patients. A significantoverlap between the two entities was not excluded at the time of diagnosis ofVAT, a situation confirmed by the authors when they approached the limitationsof the studies, namely, the difficulty in differentiating VAT and VAP by theanalysis of chest radiography,8,11 and the non-use of thoracic computedtomography.Ventilator-associated tracheobronchitis has an incidence similar to that ofVAP.,10 leading to the early use of broad spectrumantibiotic therapy.,12 The most frequently isolated agents areGram-negative bacteria, mainly includingPseudomonas aeruginosa, Klebsiella pneumoniae andEscherichia coli. Equally relevant is the prevalence ofStaphylococcus aureus despite significant variation betweendifferent intensive care units (ICU).The prevalence of multiresistant agents implicated in VAT ishigh,,11,13-15 possibly becauseit hampers ventilatory weaning and extubation rather than thesubsequent development of pneumonia. Given that heavily colonizedpatients with no infection in clinical examination did not present significantdifferences in these variables, VAT should be considered an independentclinical entity. According to Nseir, VAT significantlyincreases mortality in medical patients possibly due to subsequent evolution toVAP, but this finding has not been confirmed by otherauthors.,9Ventilator-associated tracheobronchitis increases the duration of mechanicalventilation and the length of stay in the ICU and hospital advocate that acolonization process will occur from the established equilibrium between thebacterial virulence factors and the host defense mechanisms, which may evolve insome cases to VAT with purulent bronchiolitis associated with high counting ofcolonies or to VAP with alveolar pulmonary lesion. In addition,postmortem studies suggest the existence of a continuumbetween the tracheobronchial and pneumonic processes.Tracheal intubation facilitates the entry of bacteria into the lower respiratorytract and subsequent colonization. Some authors,3,6,12 as a strategy that allows a better diagnosticdefinition of the VAT. In this context, several studies address the importanceof semiquantitative and quantitative cultures, namely, regarding thedifferentiation between heavily colonized patients and those withtracheobronchitis or pneumonia. No consensus exists regarding the referencevalues that should be used.,6,18 However, only patients with bacterial growth≥ 105CFU/mL in the ETA have been included in the lastpublished studies,,8,10,11 and a good correlation exists between thisthreshold and semiquantitative cultures with moderategrowth. However, there are several centers wherequantitative or semiquantitative cultures are not routinely used as an integralcomponent of the diagnosis of VAT, a situation that may lead to anoverdiagnosis of this entity and that limits the extrapolation of results fromclinical trials, most of which involve patient selection based on quantitativerespiratory samples.The ETA collection for semiquantitative or quantitative culture examination issuggested by some authors and thoracic computed tomography cansignificantly increase diagnostic sensitivity by approximately44%. However, in a recently published multicentersurvey, 49.2% of the participating centers reportednever considering this possibility. Considering the frequent overlap ofmicrobiological criteria in samples collected by ETA, the collection ofrespiratory samples by bronchoalveolar lavage (BAL) or protected bronchial brush(PBB) may also be important in the differentiation between VAT and VAP, namely,by not meeting criteria for the latter entity . The non-use of antibiotics in patients withnegative quantitative cultures collected by these methods appears to besafe.-24One of the major difficulties in the diagnosis of VAT is the exclusion of a newpulmonary infiltrate and consequently the differentiation of VAP. Previousstudies have demonstrated that only 68% of VAP can be diagnosed by chest X-rayanalysis,,26 and appears to increase diagnosticsensitivity in patients with clinical symptoms consistent with pneumonia andradiography of the chest not suggestive of this diagnosis. In a prospectivestudy including 99 patients with clinic symptoms and chest X-ray suggestive ofVAP, Mongodi et al. evaluated the utility of TUS in its earlydiagnosis. They concluded that the presence of subpleural consolidations and thepresence of dynamic air bronchogram are ultrasound signals that exhibit highsensitivity and specificity for the diagnosis of VAP confirmed by bacterialgrowth ≥ 104CFU /mL in BAL or by clinical criteria if BAL isnegative under antibiotic therapy. In this context, TUS can help differentiatepneumonic from non-pneumonic infiltrates. However, additional studies are neededto confirm these results and clarify the usefulness of TUS in PAV/VATdifferentiation, especially in patients with a chest X-ray not suggestive ofVAP.When performed by experienced professionals, thoracic ultrasound (TUS) alsoexhibits high sensitivity and specificity in the diagnosis of communitypneumonia in non-ventilated patients,16 The assumptions in favor of this practiceare based on a possible reduction in the time of mechanical ventilation andhospitalization and more recently in the reduction of the progression toVAP.,8 It is unclear whether these occasional benefitsare derived exclusively from VAT therapy, as it is possible that patients withearly stage VAP are being treated for VAT.The institution of antibiotic therapy in VAT patients is not consensual, mainlyin relation to its potential advantages. The possibility of antibiotictherapy reducing VAP progression is reported by someauthors.,8,11 It is limited not only by the low accuracy ofthe chest X-ray in the differentiation between the two entities but also by thenon-use of other auxiliary diagnostic tests. In addition, the reduction inprogression to documented PAV did not translate into reduced time for mechanicalventilation, hospitalization or mortality.There is no evidence in the literature that the institution of antibiotic therapyin VAT patients results in a statistically significant reduction in the time ofmechanical ventilation and hospitalization despite the statistically significantreduction of the bacterial inoculum.,11 report a reduction in mortality with theinstitution of antibiotic therapy, although only in specific subgroups ofpatients or considering all-causemortality. These findings, which were not reproduced inother studies,,13,14 are controversial given that we do not findreferences in the literature suggesting that VAT conditions increase mortalityregardless of the use of antibiotic therapy.Some studies However,antibiotic therapy should be considered in VAT patients who present with septicshock and/or worsening of oxygenation and efforts should be made to excludeother differential diagnoses, such as VAP, a situation that may also permit areduced duration of antibiotic therapy.Considering the lack of clear benefits described in the literature, theassociated costs, the risk of toxicity and the potential emergence ofmultiresistant microorganisms, we understand that antibiotic therapy should notbe routinely used in patients with VAT despite its high prevalence. This view isshared by recently published clinical guidance standards. Therefore, the use of inhaled antibiotictherapy in such cases has been suggested. In fact, inhaled antibiotics,providing high concentrations in the respiratory tract with minimal systemiceffects, exhibited promising results in the reduction ofbacterial inoculum-33 with a possible reduction of the risk ofdeveloping bacterial resistance. These data should be confirmed in subsequentstudies.,34Despite increasing the time of ventilation and hospitalization, treatment withsystemic antibiotics does not seem to alter the clinical course of VAT, possiblyin relation to the low antibiotic concentration reached in the proximal upperairway.In Respiratory infections account for half of the antibiotic prescriptions in intensivecare. Although some authors recommend the use of antibiotic therapy inventilator-associated tracheobronchitis to reduce eventual progression toventilator-associated pneumonia, the methodology used does not exclude a significantdiagnostic overlap. Furthermore, its use does not lead to a statisticallysignificant reduction in mechanical ventilation time, length of hospital stay, ormortality.Considering the high prevalence of ventilator-associated tracheobronchitis and theconsequent significant antibiotic prescription associated with its routinetreatment, we suggest that antibiotic therapy should be considered only for patientswith ventilator-associated tracheobronchitis with septic shock and/or oxygenationdeficiency. In these cases, given that the probability of beingventilator-associated pneumonia is high, we suggest the use of other diagnosticauxiliary exams for its exclusion.Considering the limitations of the published studies, additional studies are neededwith differentiating criteria between the two entities that exclude a significantdiagnostic overlap to reliably evaluate the possible benefit of systemic antibiotictherapy in ventilator-associated tracheobronchitis.


Muskoxen are increasingly exposed to multiple stressors that may impact their health and fitness. We measured stress hormones in their qiviut (wooly undercoat), and found differences across seasons, years and between sexes. Qiviut cortisol is a promising tool for guiding muskox conservation in a rapidly changing Arctic. Ovibos moschatus) are increasingly subject to multiple new stressors associated with unprecedented climate change and increased anthropogenic activities across much of their range. Hair may provide a measurement of stress hormones (glucocorticoids) over periods of weeks to months. We developed a reliable method to quantify cortisol in the qiviut (wooly undercoat) of muskoxen using liquid chromatography coupled to tandem mass spectrometry. We then applied this technique to determine the natural variability in qiviut cortisol levels among 150 wild muskoxen, and to assess differences between sexes, seasons and years of collection. Qiviut samples were collected from the rump of adult muskoxen by subsistence and sport hunters in seven different locations in Nunavut and the Northwest Territories between 2013 and 2016. Results showed a high inter-individual variability in qiviut cortisol concentrations, with levels ranging from 3.5 to 48.9 pg/mg (median 11.7 pg/mg). Qiviut cortisol levels were significantly higher in males than females, and varied seasonally , and by year (levels significantly increased from 2013 to 2015). These differences may reflect distinct environmental conditions and the diverse stressors experienced, as well as physiological and/or behavioural characteristics. Quantification of qiviut cortisol may serve as a valuable tool for monitoring health and informing conservation and management efforts.Muskoxen ( Ovibos moschatus), large herbivores that reside at Arctic and subarctic latitudes, may be especially vulnerable to ongoing changes in the Arctic  are increasingly recognized to be associated with chronic stress (chronic implying the stress occurs over long periods of time such as weeks to months), and may in turn lead to reduced health, fitness, and survival in free-ranging wildlife , chronicUrsus maritimus) (Passer domesticus) (Canis lupus) (Ursus thibetanus) (Ursus arctos) , and long guard hairs that are produced continuously over several years and form the characteristic ‘skirt’ of muskoxen  develop a reliable technique for cortisol quantification in the qiviut of muskoxen using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), (ii) determine the natural variability in qiviut cortisol among wild muskoxen and (iii) assess the relationship between qiviut cortisol levels and sex, season and year of collection.Adult muskoxen harvested by subsistence and sport hunters were sampled from January 2013 to August 2016 in the Canadian Arctic near the communities of Paulatuk, Sachs Harbour and Ulukhaktok in the Northwest Territories (NWT), Kugluktuk and Cambridge Bay in Nunavut (NU), and away from communities on the Kent Peninsula and Lady Franklin Point (NU) , individual subsistence and sport hunts , and the qiviut marketing pathway (Ulukhaktok). These are regular activities in the communities and no animals were culled specifically for our study. Samples were obtained under Animal Care and Use Permit #AC13-0121, the Wildlife Research Permit #2013-035, 2014-053, 2015-068 and 2016-058 for Nunavut, and the Wildlife Research Permit #WL500098, WL500158 and WL500257 for the Northwest Territories.The timing of sample collection was directly linked to the traditional muskox harvesting seasons and samples were classified to different seasons  based on their collection date. For the purposes of this study, samples collected between January and early April were considered winter samples, those collected late July through August were classified as summer, and those collected October through to mid-December as fall Figure . Qiviut Hunters were asked to record the gender of the harvested muskoxen on a provided form. When this information was left blank or marked ‘unknown’, a skin sample was tested to determine the gender of the animal using laboratory genetic analyses that were performed within the Kyle Laboratory at Trent University, Peterborough, Ontario, Canada. DNA from each sample was extracted using the Qiagen DNAeasy Blood & Tissue Kit  following the recommended protocol supplied by the manufacturer. Gender was determined by amplification of the SRY and ZFX genes using the primers SRY-Y53-3C, SRY-Y53-3D , ZFX-P2-n = 4). For the samples from Ulukhaktok, hides were dried under ambient environmental winter conditions in the community, and then shipped to Calgary, Alberta, Canada from where they were exported for qiviut processing. In Calgary, hide samples were collected from the rump area, as per the hunter protocol. All samples were stored at −20°C until laboratory processing.Hunters were directed to sample the qiviut when skinning the animals by cutting a piece of hide measuring approximately 10 × 10 cm from the rump, near, but lateral to the base of the tail. The location of sample collection was standardized as hair cortisol levels may vary depending on the body region .Qiviut cortisol was quantified using LC–MS/MS. The use of LC–MS/MS eliminates potential issues of antibody cross-reactivity that are associated with enzyme linked immunosorbent assays (ELISAs) and has become the preferred method of hair steroid analysis in human clinical research . It was The goal in sample preparation for LC–MS/MS is to provide as clean a steroid extraction as possible. For this reason, sample preparation differed from methods using ELISAs  added to 2 l of cold tap water and gently stirred for 3 min. The soap solution was then chilled on ice and 20 ml was added to each of the Falcon tubes containing the qiviut. The qiviut was washed by vortexing for 30 s, then the soapy water was decanted and the qiviut rinsed thoroughly with cold tap water, and patted dry in a clean paper towel. As a final rinse, the qiviut was next transferred to a new Falcon tube, and 20 ml of isopropyl alcohol (IPA) pre-chilled to −20°C, was added and mixed by gentle inversion for 10 s, then decanted to waste. Qiviut was air-dried in paper towel for at least 24 h before extraction.The qiviut samples were re-weighed before being placed in 13 × 100 mm borosilicate glass test tubes, and submerged under 6 ml of methanol pre-chilled to −20°C. A 100 μl spike of bio-identical, deuterated internal standard  in water/methanol  was added, with calibrators  and three quality control pools  spiked at the same time. Samples were extracted for 20 h at 4°C in upright tubes (i.e. no vortexing or spinning). Cold supernatant was pipetted off the hair, transferred into new culture tubes, dried at 40°C under nitrogen (Techne® Sample Concentrator), and then stored, capped, at 4°C until reconstitution.Each dry sample was reconstituted with 200 μl of water/methanol , and vortexed for 30 s. The total volume was transferred to a 600 μl microcentrifuge tube and centrifuged at 14 000 rpm for 20 min at 4°C. One hundred and fifty microlitres of supernatant were immediately transferred into LC autosampler vials for subsequent analysis.r2 = 0.94, P < 0.001), and there was no significant difference between the mean qiviut cortisol levels obtained with or without SPE .Solid phase extraction (SPE) was not used as, during method development, we found a high correlation between the qiviut cortisol levels from twelve paired samples, whose analysis differed only by the addition or not of a SPE step , DP = declustering potential , EP = entrance potential , CE = collision energy  and CXP = collision cell exit potential .All samples were analysed by using an Agilent 1200 binary liquid chromatography (LC) system coupled to an AB SCIEX QTRAP® 5500 tandem mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in positive mode. LC separation was performed on an Agilent Poroshell 120 C18 column  at 45°C. The mobile phase A was water/methanol  and the mobile phase B was methanol/IPA . The 8.5 min gradient was 20–40% B (0–1.0 min), 40–60% B (1.0–5.0 min), 60–100% B (5.0–5.5 min), 100% B (5.5–6.5 min), 100–20% B (6.5–7.0 min) and held at 20% B (7.0–8.5 min). The flow rate was 0.6 ml/min and the injection volume was 20 μl. Nitrogen was used as the source, nebulizer and collision gas . Mass resolution in Q1 and Q3 was set to unit resolution. Three transitions were monitored, the cortisol quantifier (–1), and qualifier (–2), and the deuterated cortisol IS quantifier transitions. Specific MRM (Multiple Reaction Monitoring) conditions were: x weighted linear regression) covered the range from 0.25 through 250 ng/ml with r2 ≥ 0.99 in each run. The intra-assay variability was on average 8.3% and the inter-assay variability was 13.5%. Each cortisol result was divided by qiviut mass to obtain pg/mg. Duplicate quantitation was repeated on a second run, with new hair samples, whenever the coefficient of variation (CV), calculated as (standard deviation/mean) × 100 was ≥15%. The average from the two new replicates was then used for statistical analyses.Cortisol peak integration was performed using Analyst 1.5.1 software (AB SCIEX). Sample quantitation used the area ratio between the analyte peak and the matched internal standard peak. Calibration curves , year , season of collection , and interactions between year and season, sex and season, and sex and year. In all models, we included a random effect for location of sampling to account for possible differences in qiviut cortisol among locations due to, for example, slight differences in hunter methodology or intrinsic differences in qiviut cortisol among locations linked to environmental  or other location specific conditions. Qiviut cortisol levels were log-transformed to satisfy the linear model assumptions.R2) for mixed-effects models were calculated using the MuMIn library  as the ratio of (sample size)/(number of parameters) was small . To avoi library .Table 2Qiviut was sampled from 150 adult muskoxen between January 2013 and August 2016  times higher in males than in females. Season also had a significant effect, with qiviut cortisol levels in fall and winter respectively 1.57 (95% CI = 1.26–1.95) and 1.70 (95% CI = 1.37–2.13) times higher on average than that in summer. Levels in fall and winter were not significantly different. Regarding differences between years, qiviut cortisol levels were on average 1.28 times (95% CI = 1.03–1.58), 1.67 (95% CI = 1.33–2.10) and 1.60 (95% CI = 1.21–2.10) times higher in 2014, 2015 and 2016, respectively, compared to 2013. Estimated qiviut cortisol values according to sex, season and year can be found in the σ2location = 0.0015) compared to the residual variation (σ2resid = 0.16), but the conditional modes for the levels of the random effect at each location suggest slight differences in cortisol among locations that are consistent with the local muskox population trends, with qiviut cortisol concentrations that tend to be higher in declining populations on Banks and Victoria islands . By contrast, in ELISAs, four-parameter logistic calibration curves will have quantitation errors outside of the linear portion of the curve. Therefore, when using LC–MS/MS, even when cortisol is present at a very low concentration, the detection of a signal corresponding to this hormone can be attributed to it with high confidence . HoweverWe found a high variability in qiviut cortisol levels among individual muskoxen, with levels ranging from 3.5 to 48.9 pg/mg, corresponding to an almost 14-fold variation. This wide range of values is consistent with other studies, where approximately 1.5 to 40-fold variations are reported depending on the study, number of animals included and species of interest Table  and our Particularly high qiviut cortisol concentrations, greater than 28 pg/mg, were measured in four individuals. Although all samples were washed the same way, the potential remains for cryptic blood to persist on the hair surface and be extracted. Since blood concentrations of cortisol are considerably higher than hair concentrations, blood contamination could substantially increase measured results. However, these exceptionally high ‘outlier’ hides were re-tested and similar results attained, suggesting that these are real values, and not a result of undetected, local blood contamination. Moreover, these results are consistent with other studies where individuals with higher cortisol levels are commonly identified  were significantly lower in the winter than in all the other seasons (Cervus canadensis) in South Dakota, USA, FGM concentrations were at their lowest in winter and peaked in the summer, conceivably due to increased human disturbance, high temperatures, or normal seasonal metabolic patterns (Cervus elaphus) in Austria, with a significant effect of snow and minimum ambient temperature (Loxodonta africana) presumably due to less nutritious and more sparsely distributed sources of food, and an increased competition between individuals (Equus quagga) and springboks  during the dry season in relation to decreased food and water availability . For example, faecal glucocorticoid metabolite (FGM) levels in bighorn sheep (Rangifer tarandus) and reindeer (R. tarandus) by significantly reducing their time spent foraging, and by augmenting their energy expenditure through increased movements and time spent standing  and lacstanding . Despitestanding . This maPudu puda) during the rutting season (Brachyteles arachnoids hypoxanthus) (Sapajus apella) . Muskoxen react to snowmobile activity  and in tW = 65, P = 0.98) , one of the muskox’s closest taxonomic relatives, described higher FGM levels in males (Bison bison) (Diceros bicornis) and white (Ceratotherium simum) rhinoceros , exposure to pathogens, or a combination of these and other factors that could have affected both long and shorter-term stress levels. During this time period, the average annual surface air temperatures in Arctic Tundra region of Canada from 2013 to 2015 were 1°C, 1.1°C and 1.3°C above the 1961–1990 reference period, with average summer air temperatures 0.8°C, 1.1°C and 1.2°C, above the reference period (data from Environment Canada). These warm temperatures, and other associated ecological changes, may in part be contributing to increasing stress levels.All the samples collected from 2013 to winter 2015 were analysed during the summer of 2015, whereas the samples collected onwards were analysed during the subsequent summer. Possible hormone degradation during storage may, therefore, have had a confounding effect on the yearly differences we observed. However, multiple studies, including a small pilot study by our team, have shown that cortisol is highly stable over long periods of time in hair at room temperature and in other matrices  at subzero temperatures (Chlorocebus pygerythrus) hair measured using four different immunoassays , DRG International ELISA , Salimetrics ELISA  and IBL luminescence immunoassay (LIA) ) with ones measured using LC–MS/MS (P < 0.0001), but hair cortisol concentrations were 2.5 to 20 times higher when measured using immunoassays than with LC–MS/MS. The Salimetrics ELISA and IBL LIA gave the closest results to LC–MS/MS with concentrations only approximately 2.5 times higher  to leach inside the hair shaft, thereby confounding our results . Its conTamias striatus) (Lynx canadensis) (Bos taurus) (Additional research is needed to provide evidence that qiviut cortisol levels reflect adrenal activity, which will enable us to link high qiviut cortisol levels with stress. ACTH challenges involving repeated weekly injections over several weeks have been used to validate that the measurement of cortisol in hair reflects adrenal changes in several species of domestic or captive wild animals, including eastern chipmunks (Finally, the determination of the qiviut growth cycle was based on the observation of captive animals in Saskatoon, Saskatchewan, Canada. The exact timing of the qiviut growth cycle in wild muskoxen in the Arctic remains unknown, but is unlikely to differ substantially as qiviut is shed at a similar time . Qiviut The Arctic ecosystem is currently experiencing one of the greatest rates of climate and ecological change in the world . These dSupplementary DataClick here for additional data file.


The integrity of cast-in-place foundation piles is a major concern in geotechnical engineering. In this study, distributed fibre optic sensing (DFOS) cables, embedded in a pile during concreting, are used to measure the changes in concrete curing temperature profile to infer concrete cover thickness through modelling of heat transfer processes within the concrete and adjacent ground. A field trial was conducted at a high-rise building construction site in London during the construction of a 51 m long test pile. DFOS cables were attached to the reinforcement cage of the pile at four different axial directions to obtain distributed temperature change data along the pile. The monitoring data shows a clear development of concrete hydration temperature with time and the pattern of the change varies due to small changes in concrete cover. A one-dimensional axisymmetric heat transfer finite element (FE) model is used to estimate the pile geometry with depth by back analysing the DFOS data. The results show that the estimated pile diameter varies with depth in the range between 1.40 and 1.56 m for this instrumented pile. This average pile diameter profile compares well to that obtained with the standard Thermal Integrity Profiling (TIP) method. A parametric study is conducted to examine the sensitivity of concrete and soil thermal properties on estimating the pile geometry. Pile foundations have been in use for thousands of years  while coThermal integrity testing relies on measuring the amount of heat generated by a concrete element during curing. For a pile, the heat generated and its dissipation rate at a given location are a function of the concrete mix, the pile radius and the ground conditions. However, some defects such as poor quality concrete, necking, bulging, voids or soil inclusions will cause local abnormalities in temperature near the defect. Typically, an area in the concrete cover with temperature measurements lower than the overall cover average in the same ground layer would indicate a reduction in concrete volume and therefore a likely decrease in pile diameter. Inversely, higher temperature would indicate an increased concrete volume such as a bulge ,12. HencDistributed fibre optic sensing (DFOS) is gaining prominence in the field of structural health monitoring because of several advantages such as high spatial density of data, ease of installation and reliability. It has been used successfully in several applications such as monitoring the performance of ageing or new tunnels ,14,15; tThis study proposes to build on the standard thermal integrity method, or TIP, through the use of distributed fibre optics sensing as an alternative to conventional thermal probes inserted through access tubes or embedded thermal sensors . This apThis paper presents a case study of a thermal integrity test carried out on a large bored concrete pile in London using a DFOS technique known as Brillouin Optical Time Domain Reflectometry (BOTDR). BOTDR was used to derive temperature profiles along the pile during concrete curing and the data were subsequently analysed by a one-dimensional axisymmetric heat transfer finite element model. It is shown that the pile diameter profile evaluated from the thermal data and heat transfer model is in relatively good agreement with conventional TIP results. A parametric study was conducted to examine the sensitivity of the concrete and soil thermal properties in estimating the pile profile using this DFOS-based thermal integrity testing technique.The DFOS method used in this study is BOTDR, which is based on spontaneous Brillouin scattering. In general, it relies on the fact that when light travels through an optical fibre a small amount is backscattered due to small refractive-index or density fluctuations. The backscattered light spectrum has various components including the Brillouin frequency peaks, the position of which is sensitive to density changes caused by external factors such as strain and temperature.Brillouin scattering arises from the interaction of the incident light wave photons with propagating density waves or acoustic phonons. These acoustic vibrations are generated by the thermal agitation of atoms in silica fibres and lead to density and refractive-index fluctuations. As long as the amount of light that is scattered by thermal fluctuations is too small to excite further fluctuations in the density of the medium, the process is known as spontaneous Brillouin scattering . The scaThe relationship between this frequency and changes in longitudinal strain and temperature in the fibre core/cladding can be approximated by a linear function so that ,26:(2)Δeratures ,27.TheseIn most applications, since variations in either temperature or strain can cause the Brillouin frequency to change, as in Equation (1), it is necessary to distinguish between these two effects, if one measures the Brillouin frequency change alone. One common solution to this problem, when using standard BOTDR, is to use a separate temperature compensation cable placed adjacent to the strain cable. This cable is commonly of a gel-filled loose tube construction where the optical fibre is isolated from mechanical strain effects. Hence, in this loose tube cable the frequency change is a linear function of the temperature variations only:ose tube . Hence, s et al. . Hence, BOTDR is a single ended technique where a light pulse is launched into one end of an optical fibre and the power of the spontaneous Brillouin backscattering is measured from the same end using a spectrum analyser. This power is measured in the time domain, by either heterodyne detection with a coherent receiver or optical frequency discrimination method by using a rejection filter to eliminate Rayleigh scattering signal . In the The amplitude of each frequency component is measured in the time domain and therefore the spatial position, equation :(4)z=ctFor each position, the frequency an curve .One important characteristic of distributed fibre optic sensing systems based on Brillouin scattering is the spatial resolution. The spatial resolution is the smallest distance over which strain or temperature can be measured with full accuracy. It is determined by the pulse width of the incident light. Narrowing the pulse width to improve resolution has limitations because a pulse width shorter than the phonon lifetime, which is approximately 10 ns in silica fibres, will lead to a broadened Brillouin gain spectrum, a weaker Brillouin signal and a sharp drop in measurement accuracy . Hence tA BOTDR was used in this study because it had been primarily specified to measure strain during load testing but provided a rare opportunity to gather temperature data during concrete curing for a large test pile. It is important, however, to stress that there are other distributed fibre optic measuring systems that can be used to obtain temperature distribution in concrete elements, often with greater precision and spatial resolution than BOTDR. This includes Brillouin scattering based double ended systems such as Brillouin Optical Time Domain Analysis (BOTDA)  or RamanThe project considered in this paper is located on the Isle of Dogs in East London (UK). It consists of a 60 storey tall tower with a two level basement. Two London Underground (LU) running tunnels passing beneath the site about 13 m below the deepest excavation level, to which the building was allowed to transfer just minimal load resulting in a stiff piled raft solution being employed which transferred the building load to the large diameter piles either side of and between the tunnels. As the building load was effectively spanning the 11 m exclusion zone around the tunnel, column loads of up to 95 MN had to be accommodated. Such was the magnitude of the column loads that cast-in-situ bearing piles founded in the Chalk stratum with diameters up to 2.4 m and 61 m long were required. The novel nature of this pile type posed significant design and construction risk and therefore it was decided that a preliminary test pile was required to mitigate this risk.The test pile was constructed to closely match the methodology that would later be used for the working piles. As the working piles would be required to drill past the LU tunnels during engineering hours, when trains were no longer running and personnel could access the tunnels for monitoring purposes, it was decided to construct the test pile over two days. This therefore removed any necessity to reduce the shaft resistance parameters for the working piles due to the additional time required to construct the pile. The preliminary test pile was a 1.5 m diameter bored pile to a toe level of −44.5 mOD (50.9 m below ground level-bgl) and reinforcement cage diameter of 1350 mm, as shown in The pile was load tested using two No. 670 mm diameter bi-directional Osterberg load cells located around 6 m above the pile toe. The pile was tested using Osterberg cells due the large forces that were expected to be required to fail the pile (up to 87.5 MN). During the load test, the O-cells expanded bi-axially and reached a maximum force load of 30.9 MN in each direction . The pile was monitored both during concrete curing and during subsequent load testing. The analysis in this paper focuses on the temperature data collected during concrete curing. The interpretation of the bi-directional O-Cell load test data is presented in Pelecanos et al. .A BOTDR DFOS instrumentation scheme was installed alongside conventional thermal integrity testing sensors (TIP) and standard loading test instrumentation to measure temperature, strain and displacement during concrete curing and pile loading.Both strain and temperature single mode optical fibre cables to be used with a BOTDR analyser were installed on the pile. The strain cable, which consisted of a four-core reinforced ribbon cable manufactured by Fujikura , was used to measure strain during the load test and is not discussed further since this study focuses on temperature during concrete curing. A detailed description of the strain cable and its calibration is given in Kechavarzi et al. . The temThe cable was calibrated in the laboratory over a range of temperature representative of the conditions found on site to obtain the value of The temperature cable sections used for analysis in this study consisted of two cable loops installed on four opposite sides along the pile. Their position is indicated in The above procedure was repeated with the subsequent cage sections. Outside the pile, the cables were routed to a monitoring cabin where they were spliced together to form a continuous cable allowing for a single connection point to the spectrum analyser.The spectrum analyser used in this study was a Neubrescope NBX-5000 BOTDR Analyser manufactured by Neubrex . The measurement precision or repeatability of the analyser (twice the standard deviation of the noise), for the measured distance and the spatial resolution used, was specified as ±2 °C. This repeatability or precision was tested in situ over two lower sections of approximately 40 m each for cables T-3 and T-4 one month after concrete curing. This assumed that the ground temperatures at depth lower than 10 m would not change significantly over a day. The precision obtained over 30 consecutive measurements was ±2.6 °C, which is close to the specified value obtained under ideal laboratory conditions. The spatial resolution, the minimum distance over which a change in temperature need to occur to be detected with full accuracy, was 0.5 m. The sampling resolution, the distance between two data points calculated from the time interval between two consecutive sampling points digitised by the instrument, was 0.05 m. This provided data points every 0.05 m  though each of them was affected by temperature changes over the spatial resolution of 0.5 m. The acquisition time for each data set was 7 to 8 min, representing the time needed to average In addition to the distributed fibre optics, the test pile was instrumented with vibrating wire strain gauges and extensometers. This supplemented the continuous strain data provided by the fibre optics along the pile depth. These data, which broadly agreed with that provided by the DFOS method, are presented in Pelecanos et al.  and are The pile was also instrumented by the contractor with Thermal Integrity Profiling (TIP) wires, consisting of cables with thermistors installed in series, to test the integrity of the pile concrete. This method was chosen in preference to Crosshole Sonic logging to avoid the cage becoming overly congested with the required access tubes. The principle of the method is described briefly in The analysis in this paper focuses on temperature data collected during concrete curing. Concreting of the pile was carried out on 31 January 2014 between 09:25 and 14:20 by tremie placement. According to the concrete log, a total volume of 90 As described above, there are four temperature readings on every cross sections at 0.05 m intervals along the four FO cables on the shaft length and therefore close to 4000 data sets of temperature-time curves. Each temperature profile is dependent on the combination of factors of hydration heat and the heat transfer between the pile and soil, as shown in During thermal integrity testing, heat conduction is the major form of heat transfer. The fundamental law governing heat conduction for this problem is the first law of thermodynamics, commonly referred to as the principle of conservation of energy. The pile length is very large in comparison with the dimensions of the structure in the other two horizontal directions. Hence, away from the pile ends, the heat transfer is assumed to happen in the horizontal direction only. On the other hand, the thermal boundary condition at pile ends would have influence on the thermal behaviour of pile during the curing stage. As can be seen in In addition, due to the axial symmetry, the finite element model for thermal integrity testing can be simplified as a 1D model, as shown in Hydration heat plays a crucial role in the temperature development of early-age concrete ,37. It ha, b and c are the material constants controlling the distribution of hydration heat production; In this study, the hydration heat model by De Schutter  is used Schutter , as listA 1D axisymmetric finite element model with total radial length of 10 m is developed. The same FE model was used to carry out a series of back analysis to calculate the temperature changes at the four locations over all the cross section spaced at 0.05 m intervals along shaft length. The aim of this back analysis is to find the concrete cover thickness by varying the pile radius of the model (soil/pile interface position) and matching the computed temperature curve to the DFOS data at every given location. For example, the temperature curves of cable T-1 at four different depths are shown in Thermal integrity profiling (TIP) tests were carried out by means of thermal wire cables which provided another measure of the pile shape for comparison. The effective diameter profile estimated from the proposed thermal integrity testing method (black curve) by averaging the diameter data obtained with the four DFOS cables is close to the values obtained with the three TIP sensor chains (dot curve) 48 h after concrete placement, as shown in The thermal conductivities of soil and concrete vary and hence it is necessary to assess the sensitivity of this thermal property as pile radius is evaluated by conducting back analysis. A series of parametric studies is performed to investigate the effect of thermal conductivity on the performance of the proposed thermal integrity testing. As listed in In the first parametric study, the thermal conductivity of the soil is varied. As shown in In the second series of parametric study, the thermal conductivity of the pile is varied. As shown in DFOS captures early thermal data very well and is capable of being a standalone system in future installations. The use of temperature as measures of pile integrity shows potential to localise anomalies.The advantages of using DFOS to investigate integrity over other techniques are: (i) its capacity to provides a high spatial density of data and (ii) its ease of installation and (iii) a very low sensor failure rate.Finite element back analysis is able to quantify the relationship between temperature curve and pile radius. The predicted average pile diameter profile is shown to be close to that obtained with the conventional TIP results, providing some validity to the proposed thermal integrity testing and data interpreting method.The selection of thermal conductivity values for concrete and soil can have large effects on evaluating pile profile from DFOS-based temperature curve data. It is therefore necessary to have a good dataset of thermal properties for both concrete and soil in order to ensure the proposed thermal integrity testing method produces good estimate of pile geometry.The need for better integrity testing methods in bored concrete pile construction is evident from a review of literature and industry practice. Thermal integrity testing, which uses the heat generated by hydrating concrete as a measure of integrity, is a promising relatively new technique. This paper proposes the use of DFOS, or other distributed fibre optics method such as DTS, and a simple numerical model as an alternative approach to assess the integrity of the concrete cover of concrete elements. The proposed method was tested on a long pile in the field. The following conclusions are derived:


Blood. 2015;126(26):2821-31). However, the underlying mechanisms of c-MET activation within PEL cells remain largely unknown. To solve this puzzle, here we have utilized the next generation sequencing (NGS) based bioinformatics approach to investigate the genomic landscape of the c-MET gene and we found that there's no single nucleotide variations (SNVs) occurred in the c-MET genomic regions in a cohort of PEL samples. Consistently, Sanger sequencing analysis of frequently mutated exons such as exon 10, 14 and 19 shows no mutation of these c-MET exons in PEL cell-lines, which further supports the notion that mutations are not the major mechanism responsible for c-MET activation in PEL. Further, we found that a transmembrane receptor protein, Plexin-B1, is expressed in PEL cell-lines, which is required for c-MET-mediated PEL cell survival via direct protein interaction.The oncogenic Kaposi's sarcoma–associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL), which is mostly seen in immunosuppressed patients. PEL is a rapidly progressing malignancy with a median survival time of approximately 6 months even under the conventional chemotherapy. We recently report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated in PEL cells and represents a promising therapeutic target ( The DNA tumor virus, Kaposi's sarcoma-associated herpesvirus (KSHV) can cause several human cancers especially in the immunocompromised populations . One sucMet proto-oncogene, and the hepatocyte growth factor (HGF) is the only known c-MET ligand [One of receptor tyrosine kinases (RTKs), c-MET, is encoded by the T ligand , 7. SincT ligand . MultiplT ligand . PreviouT ligand –15. CandT ligand , 16; 2) T ligand ; 3) Y100T ligand .We recently showed an abnormal activation of the HGF/c-MET signaling pathway within KSHV+ PEL cell-lines . One of The human c-MET gene consists of 21 exons and encodes a protein of 1408 amino acids (aa). Structurally, the c-MET protein contains both extracellular and intracellular domains. Its extracellular domain can be further divided into the semaphoring domain, the plexin-semaphorin-integrin domain, and four immunoglobulin-plexin-transcription domains. The intracellular domain of c-MET carries its tyrosine kinase activity  having the same length of exon 10, 14 and 19 as that from A549, one of NSCLC cell-lines without c-MET missense mutations   passages.The PEL cell-line BCBL-1 (KSHV+/EBV-) was cultured as described previously . The othRaw whole exome sequencing reads from five kidney/renal carcinoma samples  and five lung adenocarcinoma samples  generated through the National Institutes of Health, The Cancer Genome Atlas (TCGA) project were obtained from the National Cancer Institute Genomic Data Commons. Raw sequencing reads from 16 PEL cell-lines were obtained from the National Center for Biotechnology Information short reads archive (accession no. SRP032975). Raw sequence data were first aligned to the human reference genome hg19 with the Burrows-Wheeler Aligner. Next, the resulting alignment files were analyzed using a detection pipeline consisting of Samtools mpileup and VarScan to identify candidate SNVs. Lastly, the identified SNVs were annotated using the ANNOVAR software and the c-MET specific SNVs were then plotted as a heatmap based on the number of reads covering that specific SNV on a log10 scale.Genomic DNA from PEL cells was extracted using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. Primers used for amplification of specific exons of c-MET gene are listed in Table Plexin-B1 ON-TARGET plus SMART pool siRNA, or negative control siRNA (n-siRNA) (Dharmacon), were delivered using the DharmaFECT transfection reagent according to the manufacturer's instructions.For RNA interference, For cell apoptosis assays, the fluorescein isothiocyanate (FITC)–Annexin V and propidium iodide (PI) Apoptosis Detection kit I (BD Pharmingen) was used as previously described . For celTotal cell lysates (20μg) were resolved by 10% SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies for cleaved caspase 3/9, cleaved PARP, phosphor (p)-c-MET/total (t)-c-MET, p-Chk1/2, Cyclin A2, Cyclin B1 , Plexin-B1 (Santa Cruz), and β-Actin (Sigma) for loading controls. Immunoreactive bands were identified using an enhanced chemiluminescence reaction (Perkin-Elmer), and visualized by autoradiography. Immunoprecipitation assays were performed using Catch and Release Immunoprecipitation Kit (Milipore) according to the manufacturer's instructions.Significance for differences between experimental and control groups was determined using the two-tailed Student's t-test (Excel 8.0), and p values <0.05 were considered significant.


Metformin showed significant dose- and time-dependent killing effects on in vitro cultured protoscoleces and metacestodes. Notably, the combination of Met together with the minimum effective concentration of ABZSO had a synergistic effect after days 3 and 12 on metacestodes and protoscoleces, respectively. Oral administration of Met (50 mg/kg/day) in E. granulosus-infected mice was highly effective in reducing the weight and number of parasite cysts, yet its combination with the lowest recommended dose of ABZ (5 mg/kg/day) was even more effective. Coincidentally, intracystic Met accumulation was higher in animals treated with both drugs compared to those administered Met alone. Furthermore, the safe plant-derived drug Met exhibited remarkable chemopreventive properties against secondary hydatidosis in mice. In conclusion, based on our experimental data, Met emerges as a promising anti-echinococcal drug as it has proven to efficiently inhibit the development and growth of the E. granulosus larval stage and its combination with ABZ may improve the current anti-parasitic therapy.Metformin (Met) is an anti-hyperglycemic and potential anti-cancer agent which may exert its anti-proliferative effects via the induction of energetic stress. In this study we investigated the  Echinococcus granulosus. Chemotherapy treatment for this disease has had limited effectiveness thus far, which is why it is a dire need to find new drugs for its treatment. In order to survive, E. granulosus must compete with the host for the same metabolic resources. However, the cell energy is a commodity that these organisms cannot directly obtain from the host. Therefore, our treatment strategy was to interfere with the energy-generating and cell proliferation mechanisms in the larval stage of this cestode. In this study we focus on metformin, an anti-hyperglycemic and anti-proliferative drug, which exhibits considerable in vitro and in vivo activity against E. granulosus metacestodes. In experimentally infected mice, metformin-chemopreventive effect was evidenced and the combination of this drug with low doses of albendazole improved the anti-parasitic therapy results in late stage of cyst development. Therefore, the effects of these two drugs against E. granulosus warrant further investigations, in comparison to the current monotherapy of choice in humans carried out with albendazole.Cystic echinococcosis is a worldwide zoonosis of public health concern and economic significance caused by infection with the larval stage of the cestode  Echinococcus granulosus IVTE[W/F][D/N]LVC) were also identified together with the major facilitator superfamily (-MFS- 13-residue between TMDs 2 and 3: G-[RKPATY]-L-[GAS]-[DN]-[RK]-[FY]-G-R-[RKP]-[LIVGST]-[LIM]) and signature sequences after TMDs 10 and 11 as well   .E. granulosus . Th. ThKa = ng et al . Furtheranulosus  and thatanulosus , our finE. granulosus could be, at least partly, mediated by the AMPK-TORC1 pathway [Echinococcus germinal cells to cope with the high demand of both energy and intermediate metabolites under limited oxygen supply [Trichinella spiralis [E. granulosus given that our preliminary study on the degree of fibrosis in the liver of infected mice was not enough to conclude on this point.In order to know the potential of Met to prevent a secondary hydatidosis caused by the release of protoscoleces during the surgery or the spontaneous cystic rupture, we carried out chemoprophylaxis assays. Met showed a remarkable inhibitory effect on cyst development . In the  pathway . Additio pathway , a stratn supply . This men supply . Besidesn supply . This isn supply , 70, 71.spiralis . FurtherFor clinical practice during long term treatment, Met meets the necessary criteria of a good candidate as chemopreventive and therapeutic agent such as safety, good compliance, cost-effectiveness and a clear mechanism of action . Its mosS1 FigRepresentative SEM  and TEM  images of hydatid cysts recovered from untreated mice (a-d) or treated with Met (e-h), ABZ (i-l) and ABZ+Met (m-p). ll, laminated layer; mt, microtriches; dc, distal cytoplasm; gl, germinal layer; ve: vesicles (double-headed arrow); gly, glycogen storage; cc: calcareous corpuscles. Bars indicate: 50 μm in , 20 μm in , 10 μm in , and 1 μm in .(TIF)Click here for additional data file.S2 FigRepresentative SEM  and TEM  images of hydatid cysts recovered from untreated control mice (a-d) compared with Met-treated mice (e-h). ll, laminated layer; mt, microtriches; mit: mitochondria; dc, distal cytoplasm; gl, germinal layer; nu, nucleus; ly, lysosomes; gly, glycogen storage; al, autophagolysosome; Bars indicate: 20 μm in , 10 μm in  and 1 μm in .(TIF)Click here for additional data file.S3 Fig(A) Histological examination of liver tissues from 6 months E. granulosus-infected mice after oral administration of vehicle (C), metformin , albendazole  and their combination  during 60 days. The inflammatory response (double black arrowhead) was assessed by hematoxylin and eosin (H&E) staining (a-d), while fibrosis (double black arrowhead) was assessed using Masson´s trichrome (Masson TC) staining (e-h). (B) Histological examination of liver tissues from 4 months E. granulosus-infected mice after oral administration of vehicle (C) and metformin  during 60 days. The inflammatory response (double black arrowhead) was assessed by hematoxylin and eosin (H&E) staining  while fibrosis (double black arrowhead) was assessed using Masson´s trichrome (Masson TC) staining . Although the treatment with Met apparently reduced the inflammatory response and the hepatic fibrosis around of cyst, statistically significant differences were not detected in comparison with the control . Since the distribution, the development stages as well as the number of hepatic cysts were different among treatments (C and ABZ treatment showed higher number of hepatic cysts compared to Met and Met+ABZ treatments), the degree of inflammatory response and hepatic fibrosis could not be attributed only to the treatments with the drugs.(TIF)Click here for additional data file.S4 FigE-values between the protein sequences reported for E. granulosus SLC22s in GeneDB  or in GenBank  and the sequences corresponding to SLC22A isoform 1 (organic cation transporter 1 -OCT1-) for Homo sapiens (GenBank accession number O15245) and Mus musculus (GenBank accession number O08966), SLC22A isoform 2 (OCT2) for H. sapiens (GenBank accession number O15244) and M. musculus (GenBank accession number O70577) and SLC22A isoform 4 (Organic cation/carnitine transporter 1 -OCTN1-) for H. sapiens (GenBank accession number Q9H015) and M. musculus (GenBank accession number NP_062661). The “Protein size” column indicates the total number of amino acid residues in the sequences, and the “TM Domain” column gives the number of transmembrane domains of each sequence predicted by SACS MEMSAT2 Transmembrane Prediction Program. (B) Amino acid sequence comparison between Echinococcus SLC22s and mammalian orthologs. Consensus is indicated in the last line, total (uppercase letter), partial (lowercase letter), conservative changes , absence of consensus (dots) and gaps introduced to maximize the alignment (dashes). Sequences present 12 transmembrane domains (TMD1-12) (gray boxes), conserved cysteins (black arrowheads) that could be involved in the oligomerization and glycosylation sites (black circles) between TMDs 1 and 2, conserved phosphorylation sites for protein kinases between TMDs 6 and 7 (asterisks), an ASF motif (orange box), a MSF motif (green box), and key residues identified by mutagenesis assays as very important to preserve the high transport of metformin in humans  [Mus musculus (O08966 and NP_062661); Eg, Echinococcus granulosus .(A) Comparison of e boxes) . GenBank(TIF)Click here for additional data file.S5 Figactin I (actI) was used as a loading control. Molecular sizes of amplicons are indicated with arrowheads.Reverse Transcription (RT)-PCR analysis from total RNA of protoscoleces (PTS) and metacestodes (MTC) incubated for 48 h under control conditions (C) and treated with 5 mM metformin (Met) or 2.5 μM albendazole sulphoxide (ABZSO). Amplification of Eg-(TIF)Click here for additional data file.