The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. The intrinsic fluorescence was observed above 320 nm using a cut-off filter after excitation at 280 nm. Unfolding was initiated after 11-fold dilution of native protein solved in 20 mM Na phosphate, 100 mM NaCl pH 7.4 with urea (>2.2 M) in the same buffer. For refolding experiments N-BAR was unfolded in 4 M urea for 30 min at 15°C before refolding was started by 11-fold dilution. All experiments were repeated five to eight times under identical conditions, analyzed and averaged using the program Origin 7.5. Unfolding reactions were fitted with first-order reactions with the required number of exponentials:Y(t) is the signal at time t, Y0 is the offset, Yi is the amplitude of the corresponding kinetic phase and ki is the first-order rate constant of this phase. A sum of first-order reactions and one second-order reaction was fitted to refolding kinetics:kapp is the observed apparent rate constant related to the second-order rate constant k2nd as follows:N = 4 and to reduce the number of parameters, k2 was fixed to the value from the corresponding fluorescence anisotropy experiment and k4 to the value from manual mixing. For unfolding measurements the final protein concentration was 1 μM, for refolding measurements various concentrations from 0.1 μM to 5 μM were used. The slowest refolding reaction was measured by manual mixing and analyzed according to one first-order reaction.Fitting of N = 2) as described above. The obtained amplitude plot of the unfolding phases as a function of refolding time was used as an indication for the formation of intermediates and the native state. The amplitude traces of the fast unfolding phase were fitted with N = 1) or N = 2) for the amplitudes of the slow unfolding phase.Double mixing experiments were achieved for the N assay and for the U assay in the sN = 2). The obtained amplitude plots of the refolding phases as a function of unfolding time were used as an indication of the formation of unfolded protein. The observed traces were fitted with N = 1) for the amplitudes of λ1, and N = 2) for the amplitudes of λ2 and λ3.The U assay was initiated by unfolding of 12 μM native N-BAR in a 1:1 mixing step with 9 M urea to get 6 μM protein in 4.5 M urea. Refolding was started after various unfolding times (0.1 s to 300 s) by 6-fold dilution with refolding buffer resulting in 1 μM N-BAR in a final concentration of 0.75 M urea. The kinetic traces were analyzed using N if phases were missing.Far-UV CD stopped-flow was observed at an Applied Photophysics PiStar CD stopped-flow spectrometer. Changes in the ellipticity were followed at 225 nm in a 1cm curvette. Refolding and unfolding kinetics were measured at urea concentrations as described for the fluorescence stopped-flow experiments. The protein concentration was 1μM after mixing for urea dependent measurements and 0.25 μM to 4 μM N-BAR for protein concentration dependent refolding experiments. Fitting of the single- and double-mixing experiments were obtained with Eqs N in Eqs Kinetic fluorescence anisotropy measurements were performed with an Applied Photophysics SX-20 stopped-flow spectrometer with a T-format dual channel detector system for simultaneous detection of parallel and perpendicular emission. For all fluorescence anisotropy measurements the excitation wavelength was set to 297 nm using a xenon-mercury lamp. The intrinsic fluorescence was again followed above 320 nm using a cut-off filter. The experiments were designed and analyzed in analogy to the fluorescence measurements described above with reduced 2. The Gibbs free energy for unfolding (ΔGu°) was 86.1 kJ/mol with a cooperativity factor  of 24.3 kJ/mol-1M-1, the transition midpoint is at 2.25 M urea at 1 μM N-BAR  the transition midpoint shifts to higher urea concentrations  depends on the protein concentration . At the corresponding urea concentration λ5 is only visible in interrupted refolding experiments. Below 3 M urea λ6 was measured with manual mixing  shows a weak urea dependence and increases only about 10 fold between 2.5 M and 5.5 M urea. In the same range the slow unfolding rate constant (λ6) increases by a factor of 10000. Both unfolding phases measured by fluorescence anisotropy show the same dependence in the chevron-plot (data not shown) as measured by fluorescence. The refolding phases could be determined between 0.4 M and 2.3 M urea. Below 1 M urea four refolding phases could be resolved. The rate constants for λ1 and λ4 are almost independent from the urea concentration. The slowest refolding process (λ4) is assumed to result from a peptidyl-prolyl cis/trans isomerization reaction[trans conformation in the native state according to the crystal structure of N-BAR. We tried to accelerate this very slow reaction with peptidyl-prolyl isomerases, a specific class of enzymes which catalyses this types of reactions. However, in the presence of different isomerases no significant acceleration of this reaction could be observed. λ2 shows the strongest urea dependence of all refolding events; this suggests major conformational rearrangements related to this phase. Between 0.4 M and 1 M urea this phase shows a rollover. With fluorescence anisotropy measurements we could verify this phase and thus the analysis of the single-mixing experiments. The third refolding phase λ3 is only observable between 0.4 M to 1.2 M urea and shows even a slight acceleration up to 0.7 M urea.The chevron-plot for the fluorescence detected kinetics contains further details . The fas reaction,39. It scis and trans conformations resulting in two parallel pathways[2. At this state the dimeric intermediates proceed differently: intermediates with the native trans prolyl conformation  can directly fold to the native form. Intermediates with the corresponding cis prolyl conformation  first isomerize very slowly into the native prolyl isomer before forming the dimeric native state N2,trans. To confirm this proposed folding mechanism, the now following experiments have been performed.From these results and experiments discussed below we suggest a folding mechanism for N-BAR presented in  pathways. The fir5 = 6.4 s-1 and λ6 = 0.5 s-1. The same two phases are observed in single-mixing experiments at this urea concentration (see 5) and show the build up of an intermediate state with a maximum at about 10s before it slowly decays again. The red filled symbols indicate the slow unfolding process (λ6) and its amplitudes show the generation of the final native state. Thus the progression of amplitudes of both unfolding phases reports about three different refolding steps of N-BAR. To determine during which phase the dimerization reaction happens we repeat this N assay at three different protein concentrations, because the association of monomers should be concentration dependent. Only the amplitudes of the fast unfolding process . Fourth, a very slow refolding reaction (0.01 s-1) towards the native protein leads to an increase of the amplitudes of λ6 and a decrease of the amplitudes of λ5. This shows that the formation of one fraction of native molecules is significantly retarded. Because of the low rate constant, proline isomerization most probably is the reason for this process which limits for this fraction the formation of native dimers. The opposite sign of the amplitudes of this very slow folding step  detected stopped-flow experiments Fig A in . Interes1 and report about the formation of the fully unfolded protein U. The black filled symbols represent the amplitudes of the second order reaction (λ2) and the red filled symbols of the slow refolding reaction (λ3). For short unfolding times (< 8 s) all unfolded chains fold back to the native state within 20 s because no cis/trans isomerization could happen in the unfolded state. All three amplitudes from the refolding phases show the same time course during this unfolding time window. Their calculated single-exponential rate constants are for the amplitudes from the fast refolding phase 1.06 s-1, for the middle phase 1.15 s-1 and for the slow phase 1.54 s-1. These rate constants agree within error well with the slowest unfolding phase λ6 at 4.5 M urea and show that this unfolding phase is the initial and rate-limiting unfolding step. For unfolding times longer than 8 s isomerization in U becomes significant and influences the amplitudes. The slowest refolding phase (λ4) becomes observable  is observed and the amplitude of this reaction increases single-exponentially with unfolding time and a rate constant of 1.8 s-1  is also not visible after long term unfolding and therefore has no major secondary structure changes are expected for this phase.To investigate, during which of the so far described phases major changes of the secondary structure occur, we measured refolding and unfolding kinetics by stopped-flow far-UV CD. Interestingly, both refolding and unfolding kinetics contained only one observable phase . Refoldintration , suggest2 (refolding) and λ5 (unfolding) detected by fluorescence changes. Confirmation of a burst phase event is given by the amplitude of the folding kinetics, where CD signals were plotted against the corresponding urea concentration  and the fixation of the chromophore towards the non-isotropically tumbling framework of the forming N-BAR (fluorescence anisotropy). Our proposed folding  represenon-pathway intermediate, most probably corresponding to Itrans/cis, where the formation of the intermediate is fast compared to the following rate-limiting dimerization reaction, which has a higher energy barrier than the formation of the intermediate. The presence of an on-pathway intermediate can be best verified by a lag-phase for the following reaction (λ2) in a double-mixing experiments, if the consecutive steps have about the same time constant. We did not observe a lag-phase for the built up of the dimeric state probably because formation of this state is more than 20fold slower compared to the formation of the monomeric intermediate. The existence of an intermediate is also underlined by the start-end value analysis which shows a loss in the amplitudes at low urea concentrations and an early accumulation of the intermediate in the inaccessible time window of a stopped-flow experiments[trans/cis is of monomeric character because the ratio of the start and end values does not change with increasing protein concentrations[trans/cis via λ1 could be directly detected only by the more sensitive fluorescence probe. Thus, with these far-UV CD experiments we can only see one part of our complete folding mechanism. We address the dimerization reaction to the main refolding event because it is observable with every probe used in this study.The simple two-state model which was observed in equilibrium unfolding curves cannot sufficiently explain our kinetic measurements because such a model predicts on single-exponential kinetics and a V-shaped chevron plot independent from the observable. In far-periments,52. Itrantrations,15. Formtrans- and cis-prolyl peptide bonds which determines the time course of quaternary structure formation, (ii) the proline isomerization does not influence the dimerization reaction and (iii) the dimerization reaction is the main refolding step regarding concomitant secondary and quaternary structure formation. Single-jump refolding kinetics using far-UV CD and fluorescence show a different number of refolding events suggesting that not all fluorescence detected refolding events are associated with secondary structure formation. The refolding phases λ3 and λ4 occur in the dimeric state which has close to native secondary structure.The measured fluorescence kinetics show the full complexity of N-BAR folding and the following properties: (i) a heterogeneous mixture of unfolded chains concerning cis/trans isomerization is not rate limiting for formation of the dimer. The fluorescence detected unfolding kinetics show single phase kinetics and only above 4.5 M urea two phase kinetics in fluorescence measurements suggesting that below 4.5 M urea the slow unfolding phase (λ6) becomes rate-limiting. Far-UV CD detected unfolding is even more sensitive to this rate limitation by λ6, because λ5 is not observable in single-jump experiments. The latter phase is only detectable in the N assay because I2,trans gets populated via the unfolded state and not only from N2,trans.The double-mixing refolding experiments monitor the same refolding events Figs  independtrans-conformation or with a non-native cis-conformation of prolyl peptide bonds in the unfolded state has been reported for other proteins[cis/trans conformation, folding starts with a very fast folding event leading to the formation of a monomeric intermediate. As mentioned we assume that the burst-phase intermediate observed in far-UV CD and the fastest observed refolding phase (λ1) observed under fluorescence detection monitor the same folding event. A direct far-UV CD signal change in single-mixing experiments corresponding to λ1 could not be resolved although the dead-time of 30 ms for this experiment is sufficient to detect a reaction with a rate constant of 25 s-1. The reason for this observation remains unclear.That folding starts from chains with the native  proteins. Indepen5 M-1·s-1at 0.4 M urea and lies three orders of magnitude below the diffusion limit of about 109 M-1·s-1. This implies that the association of the two monomers is not diffusion-limited and not only a collision event but promoted by a conformational change of the monomeric intermediates. The deviation of the observed second order rate constant from linearity above 1 μM shows that dimerization is not rate-limiting under all conditions. This suggests a change in the rate-limiting step for the dimerization above this protein concentration. It should be noted that this deviation could be seen by all three probes used here. Such a behavior was also observed for other dimeric proteins such as P22 Arc repressor or ORF56[This monomeric intermediate dimerizes independently of the prolyl conformation. The absence of a second protein concentration dependent reaction excludes a second dimerization reaction and suggests a sequential folding pathway. For other dimeric proteins including P22 Arc repressor, knotted protein YibK, and T4 fibritin, a protein concentration dependent process could also be observed and assign to the dimerization step,18,53. T2,trans is not formed by the dimerization reaction but via an additional step reported by λ3. This reaction is not present in single-jump far-UV CD and fluorescence anisotropy refolding measurements. This implies that no substantional secondary structure rearrangements occur during this phase and that the anisotropy tumbling of the fluorophores does not change. Therefore, I2,trans resembles already closely N2,trans in structural terms. However, we could show that in double mixing refolding experiments under fluorescence anisotropy and far-UV CD detection the dimerization phase could be visualized with the same rate constant as in fluorescence measurements. In these N assays this reaction nearly completely disappears with the slowest refolding reaction λ4. This also underlines that this reaction is not an off-pathway intermediate and depends on the preceding dimerization reactions.The N assay revealed that the completely folded native dimer NExtrapolation of the two fitted curve in the N assay to zero shows that the amplitudes from the fast unfolding phase are close to zero. The amplitudes from the slow unfolding reaction show that about 10% of native molecules are present at the beginning of refolding independent of the used protein concentration Figs  and 7. T3 also shows a slight acceleration with increasing urea concentration, which is typically the signature of an unfolding process. In other proteins this effect was interpreted as indication for the formation of an off-pathway intermediate[3 is not protein concentration dependent we can exclude that a non-productive dimer or higher oligomer gets formed. If an off-pathway intermediate is formed after dimerization the unfolding of this intermediate would be rate-limiting in the formation of the native state with λ3 and thus be pronounced in a lag-phase of the λ6 amplitudes in the U assay, which we do not observe. Therefore, the urea dependence of λ3 remains unclear.In contrast to the two faster refolding phases, λermediate,54. Becacis/trans isomerisation. Although this phase could not be accelerated with an isomerase, this phase has a rate-constante of 0.01 s-1, nearly no urea dependence in the chevron-plot and an activation energy of 85 kJ/mol which is characteristic for a peptidyl-prolyl cis/trans isomerisation[trans-conformation in the native state. In principle, all of the Xaa-Pro peptide bonds can produce slow isomerization reactions because in the unfolded state an equilibrium between the cis and trans conformation can evolve[cis fraction in the unfolded state in comparison to other amino acids[cis content is up to 34%. The isomerization of this peptide bound leads to a local change in the chemical environment of the preceding tryptophan and thus to a change in the fluorescence. Along these lines, the isomerization of Trp94-Pro95 is most probably the main reason for the observable slow refolding phase λ4.We assigned the slowest refolding reaction to a peptidyl-prolyl erisation. Each N-an evolve,55. The ino acids,57. For cis/trans isomerization comes from the triple-jump experiment, which prepares only Utrans before the N assay. Here no further change in the amplitudes is observable with refolding times longer than 10 s. Additionally, the amplitudes from λ5 which report the dimerization reaction do not reach the high plateau value observed in the N assay started from a cis/trans equilibrium. This result underlines that without isomerization in the unfolded state no refolding retardation by λ4 takes place and thus the entire upper folding pathway in cis is missing.A further hint that the slowest refolding reaction results from a peptidyl-prolyl cis or in all-trans. In principle mixed dimeric states could form. λ4 does not depend on the protein concentration and λ3 and λ4 give no change in the far-UV CD spectrum. Therefore, I2,cis has to be a dimer and both, I2,cis and I2,trans have native secondary structures. The latter might also prevent PPIases to reach the prolyl peptide bond for catalysis. This implies that the major structural rearrangements during refolding can occur independent from the prolyl conformation and thus mixed dimers (one cis and one trans prolyl monomer unit) should be possible. A clear answer about their appearance or absence can not be given by the presented experiments.In our proposed model we assign both dimeric states–the intermediate and native state–in all-2,trans → I2,trans as rate-limiting unfolding step. This unfolding phase is detectable with all three employed spectroscopic probes corresponding to λ6. The following unfolding phase λ5 is best pronounced in the fluorescence experiments under strong unfolding condition. Below 4.5 M urea this phase is only detectable by double-mixing experiments, which populate I2,trans via refolding and thus becoming detectable again by all three spectroscopic probes. Rate limiting λ6 is also the reason that both performed U assays (fluorescence and fluorescence anisotropy detected) only contained the slowest unfolding phase. Such a behavior was also observed for other proteins[trans/cis → Utrans/cis), which is expected from our folding model, is not visible in any experiment. We assume a very fast kinetics and/or probably a small amplitude for this final unfolding step.Unfolding follows a sequential pathway with N proteins for the In terms of the biological function of N-BAR we could show by the equilibrium studies that the N-terminal helix0 does not influence the thermodynamic stability of N-BAR, at least in the absence of membrane lipids. The radius of curvature of N-BAR is defined by kinks in the helix bundle at well conserved positions giving rise to the dimensions of membrane tubules . The herS1 FileFig A) and far-UV CD spectroscopy . The black line shows the nativ protein while the unfolded protein in 7 M urea is represented by the red line. All data were obtained at 1 μM protein in 20 mM Na phosphat, 100 mM Na chlorid, pH 7.4 and 15°C as described in Materials and Methods.Spectra were detected with fluorescence ((PDF)Click here for additional data file.S2 FileFig A) and far-UV CD . Black symbols indicate the transition curve started from native protein and red symbols show the curve from unfolded protein in 7 M urea.Urea transition curve of N-BAR detected by fluorescence ((PDF)Click here for additional data file.S3 FileFig A). The two curves show a two-state transition and superimpose very well. Comparison of the transition curves of N-BAR (black circles) and BAR (red circles) measured with fluorescence .Urea induced unfolding curve of (1–32)BAR measured with fluorescence (black circles) and circular dichroismn (red circles) ((PDF)Click here for additional data file.S4 File2SO4.Transition curves of N-BAR measured by fluorescence in the absence (black circles) and presence (red circles) of 100 mM Na(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 FileUnfolding was initiated by manual mixing in 3 M urea, 20 mM Na phosphat, 100 mM Na chlorid, pH 7.4 and 15°C at a protein concentration of 1 μM. The kinetic trace was detected at 327 nm and can be best described by a single-exponential function.(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 FileFig A). Green symbols represent the fast unfolding phase while red symbols represent the amplitudes from the slow unfolding phase. The fitted rate constants from red amplitudes are for both protein concentrations λ3 = 0.5 (±0.05) s-1 and λ4 = 0.01 (± 5·10−3) s-1 which are comparable to single-mixing and double mixing fluorescence experiments. The second order rate constant from the green symbols differ slightly from fluorescence measurements. The rate constant of the amplitude decay is the same. Amplitude plot of the fluorescence anisotropy U assay . The only observable rate constant is λ6 = 1.75 s-1 which is the same rate constant observed in single-mixing experiments measured with all three probs.Amplitude plot of the N assay measured with fluorescence anisotropy ((PDF)Click here for additional data file.S9 FileAmplitude plot of the far-UV detected N assay. Green circles show the amplitudes from the fast unfolding phase and red circles from the slow unfolding reaction. The calculated rate constants are in the same range as measured in fluorescence.(PDF)Click here for additional data file.


Activity of daily living (ADL) is an important predictor of mortality in patients with chronic obstructive pulmonary disease (COPD). Increasing ADL is important in patients with COPD and assessment of ADL is one of the best ways to evaluate the status of COPD patients.The objective of this systematic review was to provide an overview of the psychometric properties of paper and pencil instruments measuring ADL in patients with COPD.English papers published from 1980 to 2014 regarding ADL in patients with COPD were searched in Web of Science, MEDLINE, Google Scholar, Cochrane, PubMed, ProQuest, and CINAHL databases using the following keywords: “COPD”, “ADL”, “activities of daily living”, “daily activities”, “instrument”, “questionnaire”, “paper-and-pencil instruments”, and “measure”. Following the Internet search, manual search was also done to find article references.A total of 186 articles were found. Of those, 31 met the inclusion criteria. Full texts of articles meeting the inclusion criteria were studied. Consensus-based standards for the selection of health measurement instruments”(COSMIN) were used to assess the quality of the studies.Data extraction form based on research aims developed by researchers and psychometric experts, with 17 questions was used.In these articles, 14 pen and paper instruments were identified for examining ADL in patients with COPD; of which, 4 dealt directly with ADL while 9 assessed other criteria i.e. dyspnea as ADL indicator. The majority of instruments only dealt with two main dimensions of ADL: Basic Activities of Daily Living (BADL) and Instrumental Activities of Daily Living (IADL), and did not consider Advanced Activities of Daily Living (AADL), which is influenced by cultural and motivational factors.Despite several ADL instruments identified, complete psychometric processes have only been done in a few of them. Selection of the appropriate instrument should focus on the aim of the study and the target construct. Chronic obstructive pulmonary disease is a major chronic health problem throughout the world . FunctioChronic obstructive pulmonary disease is typically accompanied by dyspnea  and dyspStudies indicate that 78% of patients with COPD have dyspnea even when walking at home, and are faced with difficulties in ADLs . Insufficient physical activity is the main cause of disability, severe loss of pulmonary function, early death , anxietyMost people with COPD experience a decline in functional status, but little is known about the magnitude of decline or factors that contribute to it .One way to assess the functional status is to ask patients via an ADL questionnaire . MeasuriADLs include activities and tasks that people routinely perform in their daily life inside/outside their homes . Vriendt et al. divided ADL into 3 domains namely basic activities of daily living (BADLs) including self-care behaviors, such as dressing and bathing, instrumental activities of daily living (IADLs), such as cooking, house chores, and shopping and AADLs, including voluntary behaviors influenced by cultural and motivational factors, which indicate satisfying activities beyond personal independence. The combination of all three domains of ADL includes all the activities that a person performs in daily life .In the past decades, physical activity assessment instruments were traditionally and predominantly used in epidemiological research to measure activity as treatment outcome and an indicator of health ; but newA review of studies showed that experts and researchers have used several instruments for measuring ADL in patients with COPD. Despite the importance of ADL measurement, and international guidelines on COPD and its treatment, none suggested a method, an instrument, checklist or a questionnaire to evaluate ADL. Thus, the goal of this study was to review the existing instruments for evaluation of ADL in patients with COPD, and assess psychometrics of instruments according to the COSMIN taxonomy .Systematic Reviews. CRD guidance for undertaking reviews in health care, January 2009). According to this guideline, the first step is the development of a protocol, which consists of the main goal and a set of predetermined stages and methods to perform a systematic review  in English on ADL in patients with COPD were searched in Web of Science, MEDLINE, Google Scholar, Cochrane, PubMed, ProQuest, and CINAHL data bases using the following keywords: “Chronic obstructive pulmonary disease”, “COPD”, “activity of daily living”, “ADL”, “activities of daily living”, “day to day activities”, “daily life activities”, “daily activities”, “instrument”, “questionnaire”, “test”, “assessment”, “paper-and-pencil instruments”, and “measure”. Following the Internet search, manual search was carried out to find article references relevant to our study. Titles of relevant references were searched and full texts of articles meeting the inclusion criteria were studied.1.Articles written in English.2.Original articles describing a paper-and-pencil instrument development and validation or psychometric process to evaluate BADL, IADL, and AADL in patients with COPD.3.Any article that included instruments that examined ADL in only one limb.4.Any article that included instruments that measured ADL in other pulmonary diseases.5.Any article that included laboratory, semi-laboratory, or field instruments.6.Any article that used generic instruments to measure ADL in patients with COPD.7.Articles that used paper-and-pencil instruments to evaluate BADL, IADL, and AADL in patients with COPD.The quality of articles that met the inclusion criteria was assessed. The COSMIN checklist for assessing the methodological quality of studies on measurement was used . The forPubMed database search using the above-mentioned keywords yielded 1,463 articles, and following search in other databases, 376 articles were found. After exclusion of repeated articles, titles and abstracts of 1,424 articles were reviewed. At this stage, 1,265 articles were excluded. Ultimately, 159 articles were included in the study for full text review. Then, relevant references, whose full texts had been studied, were manually searched in various databases. Twenty-seven articles were added to the study after manual assessment of references, and the full texts of 186 articles were reviewed. After this evaluation, 155 articles were excluded.The most common reason for excluding these studies was that they were either laboratory, semi-laboratory, field tests or performance-based tests, compared two instruments, examined daily physical activity in only one limb, used generic instruments to measure ADL, investigated correlation of physical activity with other variables, investigated the effect of COPD on ADL, assessed physical activity in patients with COPD after lung transplant, or assessed instrument for diseases other than COPD.The remaining 31 articles were evaluated for extraction of data and led to identification of 13 paper-and pencil instruments for investigating ADL in COPD patients.Flowchart 1 shows article search and selection method in every stage. Of the 13 identified instruments, four instruments  were developed to examine ADL in COPD patients  were designed to measure dyspnea as an indicator of ADL (Two instruments (PFSDQ-M) and (CDLM) were designed to measure BADL, and ADL-D included only dyspnea. Most of them (n=8) combined BADL with IADL and leisure activity. The initial search of the literature revealed that researchers have classified ADL in various forms. But the classification system suggested by Vriendt  and collThe results showed that construction of ADL measuring instruments varied between studies, but because some of the instruments did not include information about their psychometric properties we could not compare them. In some instruments, patient information had not been used when creating the items . In fact, many instruments followed an unclear process in construction of items, even though using information from individuals for whom the instrument is constructed and should be completed is important. Tables In the past decades, physical activity assessment instruments were traditionally and predominantly used in epidemiological research to measure activity as the treatment outcome and an indicator of health . When thOur study results showed that, although specific instruments exist for measuring ADL in patients with COPD, some experts still measure ADL in patients using generic scales, such as the Barthel index ; even thDespite the importance of ADL measurement in COPD patients, and large number of professional scientific and international guidelines on COPD and how to treat and care for it, none of these guidelines have suggested an appropriate instrument, or checklist for measuring ADL . JanaudiAlthough the American Association of Psychology (APA) has clearly stated that “the construct that the instrument is to measure must be expressed in a theoretical framework” , our study shows that none of the existing instruments is based on a proper theoretical framework that accounts for important and effective factors in performing ADL in patients with COPD. While, in patients with COPD, ADL depends on a number of variables like symptoms of the disease, fitness, level of independence and level of need for mobility aids or other people's help. The majority of instruments do not account for these. Not establishing these instruments on a proper theoretical framework is indicative of inadequate and incorrect perception of the concept of daily physical activity in patients with COPD. One of the problems in this regard is the inability to measure changes in physical activity resulting from treatment or rehabilitation and interventions to improve physical activity in patients with COPD because the instrument cannot accurately measure the concept of daily physical activity.Physical activity is a complex construct made up of many concepts. The construct of physical activity can include the ability to perform ADL and its associated symptoms, such as pain and dyspnea. As the present study shows, in most ADL measuring instruments, the construct measured is either BADL or IADL, while according to new definitions, ADL consists of three dimensions. Therefore, the majority of instruments do not measure the third dimension of ADL (AADL), which includes “voluntary behaviors affected by cultural and motivational factors, and indicate satisfactory activities beyond personal independence” . When a Paper-and-pencil instruments should have strong psychometric features, especially in terms of content validity of their items. Content validity is indicative of how accurately the instrument can measure the construct for which it was designed. Paper-and-pencil instruments should also have high construct validity and reliability . FurtherStudy results showed that only one of the ADL instruments was designed according to cultural and lifestyle dimensions and other instruments had not considered this issue; while, clearly, people's culture and lifestyle vary in different countries, and as a result their ADL will also be different. However, the AADL dimension is affected by culture and lifestyle more than the other two dimensions.None of the instruments specified the group of patients the instrument was developed for. A strong point in paper-and-pencil instruments is that they can be used in both hospital and laboratory settings and in the patients’ real life environment. Most paper-and-pencil instruments are designed in such a way that illiterate, low literate, disabled, and frail people cannot answer them. People with mobility or comprehension problems such as patients with reading and comprehension problems cannot answer these questions either. When these people are not accounted for in designing of instruments, a proportion of the population with COPD and their right for evaluation will be neglected .Systematic Reviews. CRD guidance for undertaking reviews in health care, January 2009).The ultimate aim of all studies conducted on clinical instruments or tests should be the assessment of ADL effects on management and treatment outcomes of patients . SystemaIn conclusion, the current study revealed that psychometric properties were not complete for any of the tools, which measure ADL in patients with COPD. We recommend that a proper conceptual framework first be designed for daily physical activity in patients with COPD, followed by a paper-and-pencil instrument for measuring daily physical activity in these patients. Despite several ADL instruments identified, complete psychometric process has been done in only a few. Selection of the appropriate instrument should focus on the aim of the study and the target construct. The strength of this study was in researchers’ adherence to methodology of systematic reviews in studies that expressed how systematic reviews should be performed. Although researchers tried to adhere to the protocol in all stages of the study and gather all relevant articles in the first stage of the study, there is still a chance that a relevant tool may have been missed. Sometimes, there is a difference of opinion about the inclusion or exclusion of a tool, which takes a long time to agree upon. In such cases, researchers tried to consider the most scientifically defensible criterion in changing the protocol, and to remain faithful to the protocol in all stages of the study.The most important limitation of this systematic review was that many tools used different surrogate or substitute terms, such as the phrase “functional status”, and argued that functional status indicated level of participation of the person in activity, and that it was synonymous to “activities of daily living” [108]. However, considering that such synonymous or substitute statements were not part of the study inclusion criteria, these tools were excluded. It is recommended that a study be conducted to analyze the concept of ADL in patients with COPD, to clarify if ADL construct is synonymous or a surrogate of statements or constructs.


Since the 1990s, influenza A viruses of the H9N2 subtype have been causing infections in the poultry population around the globe. This influenza subtype is widely circulating in poultry and human cases of AI H9N2 have been sporadically reported in countries where this virus is endemic in domestic birds. The wide circulation of H9N2 viruses throughout Europe and Asia along with their ability to cause direct infection in mammals and humans, raises public health concerns. H9N2 AI was reported for the first time in Iran in 1998 and at present it is endemic in poultry. This study was carried out to evaluate the exposure to H9N2 AI viruses among poultry workers from the Fars province.100 poultry workers and 100 healthy individuals with no professional exposure to poultry took part in this study. Serum samples were tested for antibodies against two distinct H9N2 avian influenza viruses, which showed different phylogenetic clustering and important molecular differences, such as at the amino acid (aa) position 226 (Q/L) (H3 numbering), using haemagglutination inhibition (HI) and microneutralization (MN) assays.Results showed that 17 % of the poultry workers were positive for the A/chicken/Iran/10VIR/854-5/2008 virus in MN test and 12 % in HI test using the titer ≥40 as positive cut-off value. Only 2 % of the poultry workers were positive for the A/chicken/Iran/12VIR/9630/1998 virus. Seroprevalence of non exposed individuals for both H9N2 strains was below 3 % by both tests. Statistical analyses models showed that exposure to poultry significantly increases the risk of infection with H9N2 virus.The results have demonstrated that exposure to avian H9N2 viruses had occurred among poultry workers in the Fars province of Iran. Continuous surveillance programmes should be implemented to monitor the presence of avian influenza infections in humans and to evaluate their potential threat to poultry workers and public health.The online version of this article (doi:10.1186/s12985-016-0472-z) contains supplementary material, which is available to authorized users. Most emerging diseases are of zoonotic origin, with wild and domestic animals acting as natural reservoirs . GlobaliThe wide circulation of H9N2 viruses throughout Eurasia, along with their ability to cause direct infections in mammals and humans, raises public health concerns on their potential role as candidates for the next influenza pandemic . H9N2 huIn Iran, the H9N2 subtype was identified for the first time in 1998 and is still circulating in the poultry population. In the affected farms the mortality rate ranges between 20 and 60 %, although this may also be attributable to co-infections with other pathogens, such as IBV or Mycoplasma gallisepticum . In spit≥20 as positive cut-off [Exposure to H9N2 AI viruses in Iranian poultry workers was previously revealed by means of HI test, using serum titre  cut-off , 29. In  cut-off  and 7.5  cut-off . In the The HA gene phylogenetic analysis of 88 Iranian H9N2 strains collected between 1998-2014 shows that they belong to two different groups of G1 lineage, named sub-lineage A and sub-lineage B for the purpose of this study . The percentage of positivity was of 12 % for the exposed group and 2 % for the unexposed group in the HI test, while it respectively amounted to 17 % and 3 % for the exposed and unexposed groups in the MN test. Antibody prevalence to H1N1pdm 2009 was of 28 % in the exposed versus 53 % in the unexposed groups by the HI test, and 25 % versus 33 %, respectively, by the MN test. For H3N2, the prevalence was 36 % in the exposed group compared to 44 % in the unexposed group with the HI test, while it was 28 % and 24 % in the exposed and unexposed groups with the MN test. The titer distribution percentage against different viruses in different tests  virus, as revealed by the HI and MN test (p = 0.0018). In particular, the exposed and unexposed groups showed a significantly different positivity both in the HI test (9 % vs. 1 %) and in the MN test (13 % vs. 2 %). For the H3N2 virus, the professional exposure was not significantly associated to the presence of antibodies (p = 0.22). This means that in both groups the probability of testing positive is almost similar. Differently, the unexposed group showed a significantly higher number of individuals (p = 0.001) seropositive to H1N1pdm 2009. If we consider that the H1N1pdm 2009 subtype does not circulate in avian species, it can be assumed that the serological data obtained for this subtype do not depend on the exposure to poultry species; other factors may have been at the origin of the distinct immunoreactivity observed against the H1N1pdm 2009 subtype in the two groups under study.The probability to be positive against A/chicken/Iran/10VIR/854-5/2008 H9N2, H3N2 and H1N1pdm 2009 viruses in different tests was calculated. The generalized linear model for binary responses applied on the H9N2 viruses indicated that the association between professional exposure or absence of professional exposure and H9 infection was significant (Results demonstrated that exposure to avian H9N2 viruses had occurred in humans in Iran (Fars province). In particular, the analysis showed a significantly higher prevalence of neutralizing antibodies against the A/chicken/Iran/10VIR/854-5 H9N2 virus in poultry workers than in the professionally unexposed group. These results are in accordance with a previously reported study from Iran by Alizadeh et al.  confirmiThe receptor binding properties of A/chicken/Iran/12VIR/9630/1998 H9N2 (Q-226)-like viruses affect their ability to infect humans. As a consequence, the infections of this virus in the exposed population are limited. On the contrary, the A/chicken/Iran/10VIR/854-5 H9N2 (L-226) – like viruses, have increased ability to infect humans resulting in higher prevalence in the exposed population. However, it should be considered that the molecular analysis of Iranian H9N2 viruses demonstrated that those harbouring the Q-226 mutations apparently are no longer in circulation. Furthermore, one previous study investigating the serologic response in humans exposed to avian viruses concluded that the incidence of seroconversion is low and that the antibody response after mild/asymptomatic infections is short-lived . TherefoConsistently with the results discussed so far, the unexposed group counting 100 healthy subjects showed a very low percentage of positivity for H9N2 by the HI and MN tests, while the distribution of positive titres against H1N1pdm 2009 and seasonal H3N2 influenzas were similar in the exposed and in the unexposed groups.According to Stephenson et al.  the crosThe comparison between the serological results obtained using the HI and MN tests against H9N2 showed that the MN test detected more positive samples within the same group, however, the triple test results proved that the HI titer was reasonably more consistent than the MN test. In addition, different researches have reported that intra-laboratory reproducibility by MN is lower than the one resulting from the HI test , a fact In conclusion, this study has highlighted the potential of avian to human transmission of H9N2 AIVs, and indicated that poultry workers are at risk of infection. Avian transmission of H9N2 viruses in humans can increase the probability of human adaption, while genetic reassortment with other human seasonal viruses are means of generating an influenza virus with epidemic or pandemic potential. Hence, integrated medical-veterinary surveillance and research activities are essential in order to identify the emergence of new influenza viruses, assess the clinical significance of seropositivity and understand more on the mechanisms that favour the virus to cross the species barrier. In the poultry sector, surveillance and control programmes should be implemented to reduce the prevalence of H9N2 AIV in poultry population and minimize the risk of exposure in poultry operators.The analyses were conducted on the blood sera of 100 workers regularly exposed to poultry (exposed group) and of 100 individuals with no professional exposure to poultry (unexposed group). The individuals, belonging to the exposed group, were recruited among the operators of the poultry industry and of the University Poultry Veterinary Hospital, none of whom had been vaccinated against flu. In particular, blood samples were collected between September – December 2012 from 70 slaughterhouse workers, 30 poultry house workers and from 10 operators of the University Poultry Veterinary Hospital, all resident in the Fars province (southern Iran). In January 2013, 100 sera were also collected from subjects from the same province not professionally exposed to poultry. 98 % of the individuals included in the study were males, whose mean age among the poultry workers was of 32 years (ranging between 18–62). The mean age in the professionally unexposed group was of 41 years (range between 18–67). After blood collection the serum was separated, stored at –20 °C and subsequently sent to IZSVe for the serological analyses.The use of serum samples for research purposes was approved by the Veterinary University of Shiraz in compliance with the Iranian ethical principles and institutions and according to the principles expressed in the World Medical Association Declaration of Helsinki. Informed consent was obtained from all participating individuals.http://www.megasoftware.net) program (see results). In addition, sera of all individuals were tested for the presence of antibodies to seasonal A/Minnesota/11/2010 H3N2 and A/California/4/2009 H1N1pdm 2009 viruses. Positive control sera containing antibodies directed against the selected H9N2 antigens were produced in SPF chickens. In addition, a panel of different H9N2, H1N1, H3N2 chicken sera and an H1N1, H3N2 positive human sera were used as controls in the serological assays.To select representative H9 antigens to be used in the serological investigations, the HA gene nucleotide and amino acid sequences of five Iranian H9N2 AIV isolates available at IZSVe repository were analysed and compared with the Iranian sequences available in the GenBank. Subsequently, a nucleotide sequence dataset of 88 Iranian strains along with representative strains of G1 lineage were aligned and phylogenetically analysed constructing a Neighbour-Joining phylogenetic tree by the MEGA 5  and World Organisation for Animal Health (OIE) manual , 46. BriThe WHO MN test protocol was applied . The humThe WHO guidelines for vaccine evaluation suggest that a neutralizing antibody titre ≥40 indicate higher than 50 % protection against influenza A virus infection or disease. Based on this consideration, an individual with an antibody titer ≥40 was considered positive for different serotype in MN and HI tests .P-values lower than 0.10 were considered as significant [The Geometric Mean titers (GMTs) of the triple test per method (HI and MN) was calculated for each subject checked for the virus of interest. A binary variable was created to identify the positive/negative sample. An average titre lower than 40 identified negative samples, whereas an average titre higher than 40 identified positive samples. The Chi square test was used to verify the possible association between positivity and exposure, for each method and virus. The GLM  for binary responses was used to estimate the probability to have a positive reaction for each tested virus, considering simultaneously the methods (HI and MN), the groups (exposed and unexposed) and their interaction as variables. The Maximum Likelihood method was used to estimate the parameters of the model. The Type III F-tests were applied to evaluate the overall effect of specified variables in the model. nificant . SAS 9.3


Individuals looking to improve their physique may ingest thermogenic supplements for the purposes of elevating resting metabolic rate and ultimately induce fat loss. The purpose of this study was to examine the effects of a commercially available dietary supplement (containing ingredients that promote thermogenesis) on resting metabolic rate (RMR) and hemodynamic variables in a randomized, double-blind, placebo-controlled cross-over study.TM) or a placebo and repeated the RMR, HR, and BP assessments at 60, 120, and 180 minutes post-ingestion. The thermogenic ingredients contained in the dietary supplement included caffeine, green tea extract, yohimbine HCL, and other ingredients. The placebo was void of active ingredients known to elevate RMR. Approximately 1-week later, the alternative supplement was ingested and the assessments were repeated in the exact same manner. Data were analyzed via a 2-factor [2x4] within-subjects repeated measures analysis of variance (ANOVA) using SPSS version 22.0. Post-hoc tests were analyzed via paired samples t-tests. The criterion for significance was set at p ≤ 0.05. Consent to publish the results was obtained from all participants.10 female participants  volunteered to participate in this investigation. Participants underwent two different testing sessions separated by approximately 7 days. On their first visit, participants arrived to the laboratory after an overnight fast and underwent a baseline RMR, heart rate (HR), and blood pressure (BP) assessment. Following this, each participant ingested a dietary supplement  post supplementation and significant elevations in RMR  at 2 and 3-hours post ingestion  as compared to baseline RMR values. The only elevation in the placebo treatment occurred at 3-hour post supplementation (p =0.024) time point as compared to baseline RMR values. Table TM) experienced greater elevations in RMR values as compared to the placebo treatment. These elevations came with no adverse effects relative to resting heart rate and blood pressure values. Taken on a daily basis, FitMiss BurnTM supplementation may increase overall energy expenditure possibly leading to reductions in fat mass over time.The dietary supplement treatment (FitMiss Burn


Mycoplasma hyopneumoniae enhances secondary Pasteurella multocida type A infection which leads to porcine enzootic pneumonia in infected pigs. Sixteen pigs were experimentally infected with M. hyopneumoniae and then euthanized at 7, 14, 21 and 28 days post inoculation. In situ hybridization for M. hyopneumoniae DNA and Ulex europaeus agglutinin-I (UEA-I) lectin histochemistry for fucosyl glycoconjugate, was performed in serial lung sections to determine alteration of fucosyl glycoconjugate in M. hyopneumoniae-infected bronchial and bronchiolar epithelium. Bacterial overlay assay was performed to determine the affinity of P. multocida type A with L-fucose.The objective of this study was to elucidate the pathogenic mechanisms of how M. hyopneumoniae but it was negative in the unaffected parts of the lung from M. hyopneumoniae-infected pigs and in lung from negative control pigs. Colocalization of M. hyopneumoniae and UEA-I was especially prominent in the luminal surface of bronchial and bronchiolar epithelial cells in serial section of lung. The mean number of M. hyopneumoniae-positive cells correlated with the mean number of UEA-I-positive cells in lungs from infected pigs throughout the experiment. All eight P. multocida type A isolates from naturally occurring enzootic pneumonia, bound strongly at levels of 2 μg and 5 μg of L-fucose.The luminal surface of bronchial and bronchiolar epithelial cells that were stained with UEA-I always showed hybridization signals for M. hyopneumoniae increases the L-fucose composition to enhance adherence of P. multocida type A to the bronchial and bronchiolar epithelial cells.The results of the present study demonstrate that  Mycoplasma hyopneumoniae, is characterized by dry cough, retardation of growth, poor feeding efficiency, and susceptibility of pigs to secondary bacterial infection, especially Pasteurella multocida type A [P. multocida type A is the most common secondary pathogen in M. hyopneumoniae-infected pigs, experimental infection of P. multocida type A alone is not sufficient in inducing pneumonia and generally is asymptomatic in pigs [M. hyopneumoniae exacerbates mycoplasmal pneumonia which can lead to porcine enzootic pneumonia in pigs [M. hyopneumoniae infection are the main factors underlying the secondary P. multocida type A infection [P. multocida type A infection by M. hyopneumoniae have not been elucidated.Mycoplasmal pneumonia, caused by a type A , 2. Alth in pigs , 4. In c in pigs . The damnfection , 2. NoneP. multocida type A infection in the lung. It has been reported that infection with M. hyopneumoniae enhances Ulex europaeus agglutinin-I (UEA-I), which has affinity of glycoconjugates for L-fucose residues, in ciliated epithelium of the respiratory tract in infected pigs [M. hyopneumoniae infection can alter the composition of glycoconjugates to render the lungs susceptible to P. multocida type A infection.Bacterial adherence is an important initial step in the infection process that involves specific interaction between bacterial adhesins and host receptors , 7. A vated pigs . These rM. hyopneumoniae enhances the secondary P. multocida type A infection, first, the composition of fucosyl glycoconjugates in M. hyopneumoniae-infected bronchial and bronchiolar epithelium was examined, using in situ hybridization for M. hyopneumoniae DNA and UEA-I lectin histochemistry for fucosyl glycoconjugates. Second, the affinity of P. multocida type A for L-fucose was assessed using bacterial overlay assay.In order to better understand the pathogenic mechanisms of how M. hyopneumoniae-free commercial farm based on serological testing of the breeding herd, and long term clinical and slaughter history. All piglets were negative for porcine circovirus type 2 , and PRRSV, swine influenza virus and M. hyopneumoniae  according to routine serological testing.A total of 32 colostrum-fed, cross-bred, conventional piglets were purchased at 14 days of age from a porcine reproductive and respiratory syndrome virus (PRRSV)- and n = 10 per group) using the random number generation function in Excel . At 21 days of age , 16 pigs in the infected group were intratracheally administered a 10-ml dose of a lung homogenate of M. hyopneumoniae strain SNU98703 (1:100 dilution in Friis medium) at a final concentration of 104-105 color-changing units (CCU)/ml, as previously described [M. hyopneumoniae strain SNU98703. Sixteen control pigs were exposed in the same manner to uninfected Friis medium. Four pigs from each group were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution at 7, 14, 21, and 28 dpi as previously described [Pigs aged 14 days were randomly allocated into infected or control groups (escribed . No bactescribed . TissuesA 520-base-pair DNA fragment was used as a probe. The forward and reverse primers were 5’-GTGTATCAAAATTGCCAATC-3’ (nucleotides 851 to 870) and 5’-TCCCATAACCTTGTCTTCAG-3’ (nucleotides 1351 to 1370), respectively [in situ hybridization (ISH) using a M. hyopneumoniae probe with and without DNase A treatment, one for lectin histochemistry using an UEA-I lectin, and one for haematoxylin and eosin (HE) staining. ISH was performed as previously described [M. hyopneumoiniae were used as positive controls for ISH [Tissues were routinely fixed for 24 h in 10% neutral buffered formalin. After fixation, the tissues from each pig were dehydrated through a graded series of alcohol solutions and a xylene step and embedded in paraffin wax. Four serial sections (4 μm) were then prepared from each tissue, two being further processed for escribed . The lun for ISH .Escherichia coli-infected pigs were used as positive control for lectin histochemistry of UEA-I [Sections were deparaffinized in xylene, hydrated through a graded series of alcohols to straight distilled water. Endogenous alkaline phosphatase was quenched with glacial acetic acid 20% for 2 min at 4 °C. Sections were treated for 1 h at room temperature with UEA-I lectin  at a concentration of 0.8 μg/ml in phosphate buffered saline . The sections were washed three times with PBS. The sections were then immersed in labeled streptavidin-biotin (LSAB) plus alkaline phosphatase (AP) link universal  and incubated for 15 min at room temperature. Sections were then equilibrated with Tris-buffer (pH 9.5) for 5 min at room temperature. The final reaction was produced by immersing the sections in a solution of red substrate  for 20 min at room temperature. The sections were lightly counterstained with Mayer’s haematoxylin, dehydrated through graded concentrations of ethanol and xylene, and mounted. Lectin binding specificity was tested with the following: (i) mixing the lectin with a 0.1M solution of its inhibitory sugar (L-fucose) for 20 min before performance of lectin histochemistry; and (ii) treating sections with 1 % sodium periodate prior to labeling for 10 min. Both pretreatments prevented staining. The small intestine tissues from of UEA-I . The porof UEA-I .in situ hybridization and lectin histochemistry, 3 sections were cut from each of three blocks of tissue from lung of each pig. The slides were analyzed using the NIH Image J 1.43m program (http://image.nih.gov/ij/download.html) to obtain the quantitative data. For the analysis of M. hyopneumoniae in situ hybridization, 10 fields were randomly selected and slides were scored ranging from 0  to 3  as previously described [For the morphometric analyses of escribed . For theP. multocida type A isolates from porcine enzootic pneumonia were used for radiolabeling. Radioiodination of P. multocida type A was carried out as described previously [8-109 cells in 0.3 M sodium phosphate buffer, pH 6.8) were transferred to 10 x 75 mm tubes previously coated with 100 μg of Iodogen , and reacted with 1 mCi of Na125I  at 4 °C for 10 min followed by a 5 min incubation at room temperature. Iodination was terminated by removing the cells followed by centrifugation, followed by three washes with 0.05 M Tris–HCl (pH 7.8) containing 0.15 M NaCl and 1% bovine serum albumin (TBS-BSA). The labeled bacteria were resuspended at a cell density of 5 x 107-108 cells per ml in TBS-BSA.Eight eviously  with sliP. multocida type A binding to L-fucose  was tested as previously described with slight modification [125I-labeled bacteria (approximately 1 x 106 colony forming units/ml) in TBS-BSA. Plates were washed five times with PBS to remove unbound bacteria, dried, and exposed overnight to X-ray film  at room temperature.fication . L-fucosin situ hybridization (M. hyopneumoniae) and lectin histochemistry (UEA-I). A value of P < 0.05 was considered significant.Spearman’s correlation was used to assess the relationship between in situ hybridization. The signal intensity varied within and between histological, bronchi and bronchioles, in sections of a single animal and also between pigs. Positive cells typically exhibited a dark brown reaction protduct without background staining. M. hyopneumoniae DNA was detected at the luminal surface of bronchial and bronchiolar epithelial cells , 14 (r = 0.522 and P = 0.001), 21 (r = 0.259 and P = 0.004), and 28 (r = 0.304 and P = 0.001) dpi.There was close cell-to-cell correlation when serial sections were examined by iae Fig.  and UEA-iae Fig.  was espeiae Fig.  always siae Fig.  but it wP. multocida type A isolates were analyzed for their ability to bind L-fucose using bacterial overlay assay. As shown by autoradiogram, none of eight P. multocida type A bound to L-fucose at a level of 0.5 μg. Two P. multocida type A did not bind to L-fucose at a level of 1 μg whereas the remaining six P. multocida type A bound faintly. All eight P. multocida type A bound strongly at levels of 2 μg and 5 μg of L-fucose (Fig. Eight ose Fig. . No bindM. hyopneumoniae-infected pigs enhance adherence of P. multocida type A in the bronchial and bronchiolar epithelial cells. The numbers of M. hyopneumoniae-positive cells is correlated with the numbers of UEA-I-positive cells in infected cells. UEA-I staining could also be influenced by mycoplasmal enzymes; however, M. hyopneumoniae lack the enzyme fucosidase [M. hyopneumoniae-infected pigs. In addition, eight P. multocida type A isolated from porcine enzootic pneumonia also showed strong affinity for L-fucose by bacterial overlay assay. A bacterial overlay assay was used to evaluate the binding of respiratory pathogens to glycoconjugates or glycolipids [M. hyopneumoniae infection may provide greater numbers of binding sites for P. multocida type A and subsequent infection with P. multocida type A results in severe enzootic pneumonia.The results of the present study demonstrate that increased the L-fucosyl composition in cosidase . Therefocolipids , 19. TheM. hyopneumoniae-infected pigs is potential factor to enhance colonization of P. multocida type A in bronchial and bronchiolar epithelial cells. Alternatively, M. hyopneumoniae colonizes the ciliated epithelial cells of the respiratory tract and damages the cells which lead to an enhanced infection of P. multocida type A [M. hyopneumoniae was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells. Our results are consistent with an ultrastructural study, in which M. hyopneumoniae attaches to the cilia in the bronchial and bronchiolar epithelial cells [M. hyopneumoniae is intimately associated with the cilia and causes extensive loss of these structures. M. hyopneumoniae causes ciliostasis and damages to the ciliated epithelial cells of the respiratory tract [P. multocida type A colonization and infection.Increased fucosyl glycoconjugate in a type A . A stronal cells , 22. Thery tract , 21, 22,M. hyopneumoniae enhance the secondary infection of P. multocida type A leading to enzootic pneumonia. Altered composition of glycoconjugates as the result of mycoplasma infection may be one factor that predisposes pigs to enhance P. multocida type A infection in the lung. However, co-infection with M. hyopneumoniae and P. multocida type A was not conducted in this study. Therefore, further studies are needed to confirm enhanced adherence of P. multocida type A to ciliated epithelial cells of the respiratory tract using animal model with co-infection. Altered composition of glycoconjugates by M. hyopneumoniae, together with damaging to ciliated epithelial cells of the respiratory cells, generates a favorable environment promoting colonization and secondary infection of P. multocida type A resulting in porcine enzootic pneumonia.This study explains how M. hyopneumoniae enhances secondary P. multocida type A infection which leads to porcine enzootic pneumonia in infected pigs. Altered composition of glycoconjugates by M. hyopneumoniae, together with damaging to ciliated epithelial cells of the respiratory cells, generates a favorable environment promoting colonization and secondary infection of P. multocida type A resulting in porcine enzootic pneumonia.The objective of this study is to elucidate the pathogenic mechanisms of how


Phaseonium is a three-level Λ quantum system, in which a coherent microwave and an optical control (pump) beams can be used to actively modulate the dielectric response. Here we propose a new metamaterial structure comprising of a periodic array of triangular phaseonium metamolecules arranged as a trefoil. We present a computational study of the spatial distribution of magnetic and electric fields of the probe light and the corresponding transmission and reflection, for various parameters of the optical and microwave beams. For specific values of the probing frequencies and control fields, the phaseonium can display either metallic or dielectric optical response. We find that, in the metallic regime, the phaseonium metamaterial structure supports extremely large transmission, with optical amplification at large enough intensity of the microwave thanks to strong surface plasmon coupling; while, in the dielectric regime without microwave excitation, the transmission bandwidth can be tuned by varying the control beam intensity. Implementation of such phaseonium metamaterial structure in solid-state systems, such as patterned crystals doped with rare-earth elements or dielectric matrices embedded with quantum dots, could enable a new class of actively tunable quantum metamaterials. Metamaterials are artificially engineered photonic structures displaying special optical properties, such as negative refractive index15810111215Laser-controlled quantum media in which the optical properties can be tailored by coherent excitations may be realized out of phaseonium. The term phaseonium refers to quantum coherent materials comprising of three level, atomic-like systems, where electromagnetic transparency can be induced by a control laser8Two different coherent light sources, a coherent optical light (laser) and a coherent microwave light (maser), are used to control the optical properties of the phaseonium metamolecules, while a probe light with different frequency is tuned to map its spectrum. The tuning parameters are the control laser and maser Rabi frequencies and the incident probe angle. We show that, independently of the tuning parameters, the phaseonium metamaterial displays both metallic  and dielectric  behavior at different probe frequencies. In the metallic regime, the excitation of localized surface plasmon polaritons at the sharp edges of the corrugated structure leads to a unique field distribution with nearly vanishing transmission. Only around the phase matching frequency, where plasmons coupled between different metamolecules are excited, light transmission becomes possible. In the dielectric regime, light is strongly localized in the structure, particularly its magnetic field component; trapping slows light down, and the real part of the effective refractive index increases significantly. For large microwave intensities, the dielectric phaseonium metamaterial acquires unusual characteristic of a metal with gain, amplifying both transmission and reflection of the probe. This may enable unique applications in controllable optical/plasmonic devices for telecommunication and optical data processing.a, arranged in a symmetrical trefoil as indicated by the blue rectangular box in x-direction and 2a in the y-direction. In our simulations, the unit cell is repeated four times in the x-direction and periodic boundary conditions are assumed in the y-direction. The scaling relationship between the size of the unit cell and the incident probe wavelength, λ, is given by a = 2λ/3. This asymmetric structure is designed to support light trapping and guiding within the periodic array, while minimizing reflection when light propagates from left to right. In the regime where phaseonium is dielectric, the trefoil metamaterial behaves as a dielectric waveguide. Multiple reflections and interference between metamolecules limit transmission bandwidth, so that the spectral width of transmission window can be tuned (from narrow to broad) simply by adjusting the control beam intensity. Moreover, the sharp edges of triangular metamolecules induce strong plasmonic field enhancement and strong surface plasmon coupling, which enhance light transmission when phaseonium is set to the metallic state.z-direction (perpendicular to the plane of the metamolecules), while the probe light illuminates the structure in the x−y plane, with an incident angle θ with respect to the x-axis. We treat the phaseonium as a three-level quantum system in Λ configuration, as shown in ħωac and ħωab are the energy difference between levels vp and electric dipole Rabi frequency Ωp. The vc  and vm (in the microwave domain) and Rabi frequencies Ωc and Ωm, respectively. The control beam is on resonance for a stationary atom with ωac = vc, while the probe is detuned by δω = ωab − vp around stationary resonance frequency ωab of the As shown in c/γac = 40 with decay rate γac = γab = 107 s−1, and we consider two cases: without microwave excitation (Ωm/γac = 0), and with microwave excitation (Ωm/γac = 0.1). Without microwave excitation (δω/γac = ±40 (δω/γac > −5 (dielectric regime) and negative for −40 < δω/γac < −5 . When the microwave excitation is turned on (m (in our case Ωm/γac = 0.1) the system shows gain when δω/γac > 0, the real part of the permittivity is negative for broad range −40 < δω/γac < 40, while the imaginary part of the permittivity become negative near δω = 0  to metallic-like .The permittivity of phaseonium can be tuned by varying the Rabi frequencies of the citation , the sysac = ±40  and is aac = ±40  is positurned on , the EITT and reflection R spectra of the structure and the corresponding spatial distribution of the electric and magnetic fields with various input conditions. Since without microwave irradiation the imaginary part of the phaseonium refractive index is always positive, the metamaterial is expected to behave as a lossy waveguide.We firstly focus on the basic scheme in which the microwave beam is absent to investigate EIT of the probe field for different strengths of the control beam. We investigate the transmission T and R spectra of the structure as a function of Ωc when the probe beam illuminates the structure from left to right (L-R) and right to left (R-L), respectively. Here we assume that the periodic trefoil array is surrounded by air. δω, and one where transmission is significantly lower (large reflection) at large δω. Notably, the transmission bandwidth can be tuned by changing the intensity of the control beam, with a wider transmission window for larger control beam intensity. In the center region of high transmission, the phaseonium optical response is dielectric  > 0 for δω/γac > −5), while in the lossy regions where Real (ε) < 0 (−40 < δω/γac < −5), phaseonium is metallic. Nevertheless, even in the metallic regime the phaseonium metamaterial displays a secondary transmission peak at negative detuning, whose frequency depends on the control beam intensity.δω = −27.59 , δω = −15.23 , δω = 0.75  and δω = 21.58 , while the control Rabi frequency was set to Ωc/γac = 40. In the detuning range −40 < δω/γac < −5, the real part of the permittivity is negative, and the phaseonium behaves like a metal. The structure would then support localized surface plasmon polaritons on the corrugated edges of the triangular metamolecules. A closer look to the field distributions reveals that very strong electric fields are trapped at the vertices of the triangles, strongly limiting light transmission. Interestingly, when the detuning achieves phase matching conditions, such as at frequency δω = −27.59 (A), coupled surface plasmons can be excited between different metamolecules, thus enabling transmission even when phaseonium is metallic. This explains the existence of the transmission sideband in the metallic regime , phase matching conditions are not satisfied, and the transmission vanishes. Here, energy is localized at the vertices of the plasmonic triangular metamolecule closest to the input, creating hot spots. When δω/γac > 0, phaseonium is in the dielectric regime and large transmission can be obtained, as seen in the field distributions at frequency δω = 0.75 (C). At this frequency, the phaseonium metamaterial can confine and guide light similar to a dielectric waveguide.To further understand the origin of the primary and secondary transmission peaks, we present in gime see , and theδω = 21.58 (D) with appearance of reflectance peaks. Typically, in free space, large electric field amplitudes E are accompanied by large magnetic field amplitudes B. However, in the phaseonium structure, fields can be either strongly electric or magnetic, as shown in the field distribution at δω = 21.58 (D), where the electric field in the triangle nearest to the incident beam is much stronger than the magnetic field.Transmission drops drastically at large positive detunings, particularly at δω = −15.23 (B) the R-L reflection is around 80%, while the L-R reflection is only about 20%.As seen in c) in the basic scheme without microwave excitation. m/γac varying from 0.02 to 0.2 while keeping Ωc/γac = 40 constant. Here the probe light is in transverse magnetic (TM) polarization. When Ωm is large enough, starting from Ωm/γac > 0.005, both transmission and reflection display gain, and the gain peaks blue shifted at larger Ωm. At large microwave Rabi frequencies, such as Ωm/γac = 0.2, extremely large gain is observed for both transmission and reflection (see the peaks at δω/γac = 50).So far, we have discussed the dependence of the reflection and transmission spectra of the phaseonium metamaterial on the control beam Rabi frequency , surface plasmon coupling is the main light propagation mechanism. This is evident from the electric and magnetic field distributions, where periodically and highly localized plasmons at the edges of the triangular metamolecules are seen in the high transmission point . The change in phase matching condition caused by the silica background leads to the disappearance of the secondary transmission band at negative detuning, observed in the case of air background  simply by adjusting the control beam intensity. This can be used for broadband optical switches controlled by amplitude modulation of the laser pump intensity. At high maser intensities, the phaseonium acquires the unique characteristic of a metal with gain. Thanks to the large coupling of surface plasmons, the periodic triangular trefoil structure supports extremely large transmission, with optical amplification. This effect could be exploited to realise optical amplifiers controlled by amplitude modulation of the maser intensity, which would overcome the rapid decay of propagating surface plasmons - the most common problem of lossy plasmonic systems. Furthermore, we showed that preferential amplification of the transmitted component of the probe in the forward direction forces propagation along the metamaterial axis, making this phaseonium metamaterial structure very robust against insertion angle. Finally, we discussed the possible implementation of phaseonium metamaterials in solid-state systems, such as patterned crystals doped with rare-earth elements or dielectric matrices embedded with quantum dots, by analysing the influence of a dielectric background on the optical response. We foresee that the actual implementation of phaseonium metamaterials may result in a new class of active quantum plasmonic devices with unique applications in telecommunications and optical data processing.c and Ωm of the external control light and microwave, respectively. According to calculations inOptical properties of the quantum coherence material (phaseonium) consisting of an ensemble of of three-level atomic-like systems can be controlled by Rabi frequencies Ωħ and ε0 are Planck constant and electric constant respectively. The decoherence functions Γin are given by Γca = γac + iΔc, Γab = γab − iΔ(ω) and Γcb = γbc + i(Δc − Δ(ω). The effective decay rates γin depend on the spontaneous emission rates Γi and defacing rates. In our simulation, we use the following parameters for quantum dot based8N = 1024 m−3. Finally, the permittivity of the phaseonium can be calculated bywhere εb is the dielectric constant of the background medium which is equal to 1 for air and 1.5 for fused silica.where y-direction.The transmission and reflection of the phaseonium structure with different probe direction and corresponding electric and magnetic field distributions, were computed by the finite element method (FEM) in the COMSOL package, using the refractive index dispersion of phaseonium deduced from How to cite this article: Nguyen, D. M. et al. Coherently Tunable Triangular Trefoil Phaseonium Metamaterial. Sci. Rep.6, 21083; doi: 10.1038/srep21083 (2016).


Prior studies have indicated a high prevalence of noise-induced hearing loss (NIHL) among Navy personnel; however, it is not clear whether this is caused by work on board. The present study aimed to assess the prevalence of hearing loss among Navy personnel in the Royal Norwegian Navy (RNoN), and to investigate whether there is an association between work on board RNoN vessels and occurrence of hearing loss.Navy personnel currently working on board RNoN vessels were recruited to complete a questionnaire on noise exposure and health followed by pure tone audiometry. Hearing loss was defined as hearing threshold levels ≥25 dB in either ear at the frequencies 3,000, 4,000 or 6,000 Hz. Hearing thresholds were adjusted for age and gender using ISO 7029.The prevalence of hearing loss among Navy personnel was 31.4 %. The work exposure variables: years of work in the Navy, years on vessel(s) in the Navy and years of sailing in the Navy were associated with reduced hearing after adjusting for age, gender and otitis as an adult. Among the work exposure variables, years of sailing in the Navy was the strongest predictor of reduced hearing, and significantly reduced hearing was found at the frequencies 1,000, 3,000 and 4,000 Hz.Our results indicate that time spent on board vessels in the RNoN is a predictor of reduced hearing. Noise-induced hearing loss (NIHL) is considered to be one of the most prevalent work-related diseases worldwide. A worldwide analysis states that 16 % of disabling hearing loss in adults is attributable to occupational noise exposure  were asked to take part. Information about the study was given in plenary by the management on board each vessel and was also provided through a written letter handed out prior to examination. The study was carried out by trained personnel at the two naval bases (Bergen and Sortland), supervised by a researcher from the University of Bergen. A total of 581 participants were examined in Bergen, while 191 were examined in Sortland.The questionnaire comprised questions regarding occupational and non-occupational factors which could possibly induce hearing loss Table . This inmax at 55 dB (31.5 Hz). The frequencies selected for audiometry were the following: 250, 500, 1,000, 2,000, 3,000, 4,000, 6,000 and 8,000 Hz. The equipment was calibrated prior to audiometry  with Brüel & Kjaer sound level meter Hand-held Analyzer Type 2250. The background noise was in accordance with ISO 8253-1  for all An automated procedure was used, but if there was uncertainty regarding measured hearing thresholds, ongoing tinnitus or former recognized hearing loss, manual audiometry was performed. Individual noise exposure within the last 16 h prior to audiometry was evaluated by a checklist that contained the following choice of statements regarding recent noise exposure: “No loud noise exposure,” “Loud area noise exposure,” “Loud workshop noise exposure” and “Other loud noise exposure.” Navy personnel who reported being highly exposed to noise the previous 16 h  and who had a hearing threshold ≥25 dB in either ear at 3,000, 4,000 or 6,000 Hz were excluded. In order to be included in the study, a new audiometry had to be conducted when they had not been exposed to loud noise the previous 16 h. Audiometry was not performed in cases of present acute airway infections with additional sinus, nose or ear affection, and testing was postponed until the participant became asymptomatic. In cases of inadequate completion of the test protocol, the results were excluded. All results were evaluated by the researcher in cooperation with an otolaryngologist, and referral to supplementary tests was made when indicated.A total of 772 gave their consent to join the study. In spite of a test regime controlling the audiometer and earphones, an unstable wire connection on the right earphone (Amplivox Audiocups) was discovered after some weeks of testing, affecting 110 measurements conducted in Bergen. In 24 of these cases, an additional audiometry was performed by qualified personnel assigned to the project. We were not able to retrieve the remaining 86 participants for an additional audiometry. Due to this technical failure as well as to insufficient compliance with the test protocol, results from a total of 167 participants had to be excluded, leaving 605 participants included in the study  and Pearson correlation coefficient (R).Participants were categorized into job categories according to working position on board. Hearing loss was defined as hearing threshold levels ≥25 dB in either ear at 3,000, 4,000 or 6,000 Hz. Continuous variables of potential determinants of hearing loss were categorized in quartiles. Log binomial regression provided relative risks (RR) with 95 % confidence intervals (CI) of hearing loss among the different determinants. Only determinants with significant impact on hearing were presented.In order to adjust for the influence of age and gender on hearing, a new variable was constructed. Based on ISO 7029 , we calcA Δ Hearing threshold <0 indicated a better hearing than according to ISO 7029, Δ Hearing threshold = 0 indicated hearing equal to ISO 7029, while Δ Hearing threshold >0 indicated poorer hearing than according to ISO 7029. This calculation was made for each frequency for both ears, and for each participant, the poorest Δ Hearing threshold of the two ears for each frequency was chosen.The association between the work exposure variables  in the Navy and years of sailing in the Navy) and Δ Hearing threshold for each frequency was analyzed by linear regression providing β and 95 % CI. The results were adjusted for otitis as an adult, which was the only variable apart from age with significant negative impact on hearing in our data. This analysis was only completed for the 522 participants tested with the Interacoustics AD226 audiometer, thus excluding those who were tested with the audiometer with +10 dB as the minimum test level. In a separate linear regression analysis, we excluded participants with prior otitis as an adult instead of adjusting for this. This analysis was completed for 453 participants tested with the Interacoustics AD226 audiometer.p < 0.05. The data were analyzed using IBM SPSS Statistics, version 21.Statistical significance was set at The study was performed in accordance with the 1964 Declaration of Helsinki and its later amendments. The study protocol was approved by the Regional Committees for Medical and Health Research Ethics, REC South East. The participants were informed about the objectives and conditions of the study and gave their informed consent. The participants received no payment for participating in the study, and they could withdraw from the study at any point. Individual data from the study could not be used as a basis for medical selection of candidates. The Royal Norwegian Navy permitted all available results on group level to be published.The study population consisted of 605 participants, of which 569 were male and 36 female. The mean age of the participants was 30 years, with a range from 19 to 62 years. A total of 190 participants (31.4 %) had hearing loss defined as hearing threshold levels ≥25 dB in either ear at 3,000, 4,000 or 6,000 Hz.The prevalence of hearing loss was significantly higher among navigators (37.0 %) and engine room personnel (38.0 %) than electricians 23.6 %) (Table 3.6 % (Tan = 605) showed that hearing loss was significantly associated with age, the work exposure variables: years of work in the Navy, years on vessel(s) in the Navy and years of sailing in the Navy, otitis as an adult, attending concerts/disco and use of Mp3 player , years on vessel(s) in the Navy  and years of sailing in the Navy . Among the 77 participants who had experienced otitis as an adult, 50.6 % had hearing loss , the hearing threshold level increased significantly for all the three work exposure variables at 1,000 and 4,000 Hz (Table n = 69), the hearing threshold level was significantly increased for the work exposure variable years of sailing in the Navy at 1,000, 3,000 and 4,000 Hz (data not shown). Years on vessel(s) in the Navy was associated with a significantly poorer hearing threshold at 4,000 Hz. Years of work in the Navy was not associated with impaired hearing thresholds in this analysis.Using the age and gender-adjusted variable Δ Hearing threshold and adjusting for otitis as an adult in a linear regression model  in the Navy and years of sailing in the Navy, as well as age and otitis as an adult. When adjusting for age, gender and otitis as an adult, higher hearing thresholds at 1,000 and 4,000 Hz were found when assessing the work exposure variables. Of the three work exposure variables, years of sailing in the Navy was the strongest predictor of hearing loss in our study and suggests that work on board RNoN vessels is detrimental to hearing.Similar results were also found when excluding participants with prior otitis as an adult. However, for years of employment and years on vessel(s) in the Navy, this association was weaker and might be explained by the smaller sample size when excluding those with prior otitis as an adult. Using Mp3 player and attending concerts/disco seemed to have a positive impact on hearing. This finding might be related to the assumption that those who listen to loud music may tolerate the noise exposure, hence not developing hearing loss. Another explanation can be that those who already have developed hearing loss give up attending concerts/disco and listening to Mp3 player in order to avoid further deterioration of hearing. However, usage of Mp3 player and attending concerts was inversely associated with age and years of employment and the observed association may therefore have been confounded. The prevalence of hearing loss was significantly higher among navigators and engine room personnel than among electricians, suggesting that the noise exposure varies with job category.Hearing loss can be classified in numerous ways, rendering comparison of hearing loss between different studies a challenge  rather than using those set by the U.S. Navy . Comprehensive noise level measurements on board Navy vessels have barely been reported; however, studies from commercial vessels have shown that noise levels in engine rooms range from around 90 to 110 dB(A) . Noise levels in the engine control room (where electricians have their work site) of ferries, cargo ships and westamarans range from around 70 to 90 dB(A) (Svendsen and Børresen The response rate in this study was high (81.4 %); however, the participation rate was only 63.8 %. This was due to the fact that data were collected in accordance with a stringent protocol. There is no reason to believe that the excluded participants differ from the ones included. We have limited information about the 149 who did not meet for examination and the 17 who did not give consent to participate; hence, we cannot rule out that these non-responders differed from the responders.Few previous studies on hearing loss among Navy personnel have provided information on confounding factors that might be responsible for hearing loss (Henselman et al. All invited personnel were informed that individual data would not be used to assess medical skillfulness, with criteria for hearing thresholds that must be fulfilled in order to be allowed work on board. Furthermore, there is no reason to believe that recorded hearing thresholds have been biased by participants striving to get a result adequate to be allowed to work on board.We chose to use ISO 7029  as a refAlthough we found coherence between years of sailing in the Navy and impaired hearing, the cross-sectional study design cannot clarify cause and effect.In the RNoN today, no definite protocol is established on how to follow up personnel with recognized hearing loss. We hope that this study, stating a high prevalence of hearing loss, will contribute to further awareness of the noise problem on board. Noise measurements and subsequent protection against high noise levels should be implemented, and a hearing conservation program should be established in order to improve working conditions on board. As the population is young, the benefit from prevention is great and hearing can still be protected and preserved.